Oligomeric compositions and methods

ABSTRACT

The activated oligomer compounds described herein are capable of forming bio-reversible covalent bonds with plasma proteins, in particular with human serum albumin. The plasma protein-oligomer complexes of the present invention exhibit enhanced cellular entry and significantly enhanced serum half-life.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/618,882 filed Oct. 13, 2004, which is incorporated herein in itsentirety.

TECHNICAL FIELD

The present invention describes activated oligomeric compounds capableof forming covalent bonds with plasma proteins, in particular with humanserum albumin; methods of treating a subject with the activatedoligomeric compounds; and methods of synthesizing the activatedoligomeric compounds.

BACKGROUND OF THE INVENTION

Recent reports in the literature describe the use of serum albumin forimproved drug delivery by binding the drug to the protein throughcovalent linkages. Using albumin-based drug delivery, antiproliferativedrugs, such as doxorubicin or methotrexate, have been shown to exhibitimproved pharmacokinetic properties and antitumor activity. (See Stehle,G.; Wunder, A.; Sinn, H.; Schrenk, H. H.; Schutt, S.; Frei, E.; Hartung,G.; Maier-Borst, W.; Heene, D. L. (1997) Pharmacokinetics ofmethotrexate-albumin conjugates in tumor-bearing rats. Anti-CancerDrugs, 8, 835-844; Stehle, G.; Wunder, A.; Schrenk, H. H.; Hartung, G.;Heene, D. L.; Sinn, H. (1999) Methotrexate-albumin conjugate causestumor growth delay in Dunning R332 HI prostate cancer-bearing rats.Anti-Cancer Drugs, 10, 405-411; Mansour, A. M., Drevs, J. et al. (2003)A New Approach for the Treatment of Malignant Melanoma: EnhancedAntitumor Efficacy of an Albumin-binding Doxorubicin Prodrug That IsCleaved by Matrix Metalloproteinase 2. Cancer Research 63, 4062-4066).

SUMMARY OF THE INVENTION

The present invention is directed to an activated oligomer which iscapable of forming a bio-reversible bond with plasma proteins, i.e. theactivated oligomer is bound to plasma proteins under plasma conditions,however become released under cellular conditions. The activatedoligomers of the present invention comprise an oligomer and an activateddisulfide moiety capable of forming bio-reversible bonds with plasmaproteins. In another embodiment of the present invention the activatedoligomer may optionally comprise a bivalent linker between the oligomerand the activated disulfide moiety. In another embodiment, the activateddisulfide moiety has the formula —S—S(O)₂-substituted or unsubstitutedC₁-C₁₂ alkyl or —S—S—C(O)O-substituted or unsubstituted C₁-C₁₂ alkyl.Preferred activated disulfide moieties are methane thiosulfonate anddithiocarbomethoxy. In further embodiments, the activated disulfide issubstituted or unsubstituted dithiopyridyl, substituted or unsubstituteddithiobenzothiazolyl, or substituted or unsubstituted dithiotetrazolyl.Preferred activated disulfides are 2-dithiopyridyl,2-dithio-3-nitropyridyl, 2-dithio-5-nitropyridyl,2-dithiobenzothiazolyl, N-(C₁-C₁₂ alkyl)-2-dithiopyridyl,2-dithiopyridyl-N-oxide, or 2-dithio-1-methyl-1H-tetrazolyl.

Another aspect of the invention is a method of treating a subject withan activated oligomer, comprising the steps (a) providing an activatedoligomer, said activated oligomer conjugated optionally with a bivalentlinking group to an activated disulfide moiety; and (b) administeringthe activated oligomer to said subject.

Also disclosed are methods of treating a subject with an activatedoligomer, the method comprising (a) providing an activated oligomer,said activated oligomer conjugated optionally with a bivalent linkinggroup to an activated disulfide moiety; (b) obtaining a biologicalsubstance from the subject; (c) contacting the biological substance withsaid activated oligomer thereby producing an oligomer-linked biologicalsubstance; and (d) administering said oligomer-linked biologicalsubstance to said subject.

Also disclosed are methods of treating a subject with an activatedoligomer, the method comprising (a) providing an activated oligomer,said activated oligomer conjugated optionally with a bivalent linkinggroup to an activated disulfide moiety; (b) contacting a biologicalsubstance with said activated oligomer thereby producing anoligomer-linked biological substance; and (c) administering saidoligomer-linked biological substance to said subject.

In one aspect of the present invention, the activated oligomer is anoligonucleotide, a peptide nucleic acid, or a morpholino nucleic acid. Amore preferred activated oligomer is an oligonucleotide. In someembodiments, the oligonucleotide comprises at least one 2′-modifiednucleotide, wherein the 2′-modification is selected from halogen,alkoxy, substituted alkoxy, amino, or substituted amino. Preferred2′-modifications are fluoro, methoxy, methoxyethoxy, O-allyl,dimethylaminooxyethoxy and amino. In some embodiments, theoligonucleotide comprises at least one phosphodiester internucleosidelinkage. In another embodiment, the oligonucleotide comprises at leastone phosphorothioate linkage.

In some embodiments, the activated oligomer is a peptide nucleic acid.In a preferred embodiment, the activated oligomer is a peptide nucleicacid of the formula:

wherein,

each Bx is independently a nucleobase;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the α-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH—,

R₅ is a carbonyl protecting group; and

n is from 2 to about 50.

In some embodiments, the activated oligonucleotide is a double strandedoligonucleotide. In a preferred embodiment, the double strandedoligonucleotide comprises a first strand and a second strand. In anotherembodiment, at least one nucleotide of the first or the second strand ofthe double-stranded oligonucleotide is 2′-modified, wherein the2′modification is selected from halogen, alkoxy, substituted alkoxy,amino or substituted amino. Preferred 2′-modifications are fluoro,methoxy, methoxyethoxy, O-allyl, dimethylaminooxyethoxy and amino. Insome embodiments, at least one oliogonucleotide of the first or thesecond strand of the double-stranded oligonucleotide comprises at leastone phosphodiester internucleoside linkage. In another embodiment, atleast one oliogonucleotide of the first or the second strand of thedouble-stranded oligonucleotide comprises at least one phosphorothioatelinkage.

In some embodiments, the activated disulfide moiety has the formula—S—S(O)_(n)—R₁, wherein

-   -   n is 0, 1, or 2; and    -   R₁ is selected from substituted or unsubstituted heterocyclic,        substituted or unsubstituted aliphatic, or —C(O)O—R₂, wherein R₂        is substituted or unsubstituted aliphatic.

In another embodiment, the activated disulfide moiety has the formula—S—S(O)₂-substituted or unsubstituted C₁-C₁₂ alkyl or—S—S—C(O)O-substituted or unsubstituted C₁-C₁₂ alkyl. Preferredactivated disulfide moieties are methane thiosulfonate anddithiocarbomethoxy. In further embodiments, the activated disulfide issubstituted or unsubstituted dithiopyridyl, substituted or unsubstituteddithiobenzothiazolyl, or substituted or unsubstituted dithiotetrazolyl.Preferred activated disulfides are 2-dithiopyridyl,2-dithio-3-nitropyridyl, 2-dithio-5-nitropyridyl,2-dithiobenzothiazolyl, N-(C₁-C₁₂ alkyl)-2-dithiopyridyl,2-dithiopyridyl-N-oxide, or 2-dithio-1-methyl-1H-tetrazolyl.

In some embodiments, the bivalent linking group is a bivalentsubstituted or unsubstituted aliphatic group. In another embodiment, thebivalent linking group has the formula -Q₁-G-Q₂-, wherein

Q₁ and Q₂ are independently absent or selected from substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted alkaryleneor —(CH₂)_(m)—O—(CH₂)_(p)—, wherein

each m and p are, independently, an integer from 1 to about 10;

G is —NH—C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —NH—C(S)—NH—, —NH—O—,NH—C(O)—O—, or —O—CH₂—C(O)—NH—.

Preferred bivalent linking groups are:

In certain embodiments of the methods of the present invention, thebiological substance is a plasma protein, wherein said plasma protein isselected from serum albumin protein, transferring protein, ferritinprotein, or immunoglobulin proteins. In preferred embodiments, theplasma proteins are purified plasma proteins or recombinant plasmaproteins. In yet a more preferred embodiment, the plasma proteins arehuman plasma proteins. In preferred embodiments of the presentinvention, the serum albumin is purified human serum albumin orrecombinant human serum albumin. In some embodiments the step ofadministering is intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion.

Another aspect of the present invention provides compounds, comprising apeptide nucleic acid (PNA), a bivalent linking group and an activateddisulfide moiety. In some embodiments, the PNA comprises between 8 and80 nucleobases. In other embodiments, the PNA comprises between 10 and50 nucleobases. In further embodiments, the PNA comprises between 15 and40 nucleobases. In a preferred embodiment, the PNA comprises between 15and 25 nucleobases.

In some embodiments, the PNA is of the formula:

wherein

each Bx is independently a nucleobase;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the ω-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH—;

R₅ is a carbonyl protecting group; and

n is from 2 to about 50.

In some embodiments, the bivalent linking group of the oligomericcompound is a bivalent substituted or unsubstituted aliphatic group. Inanother embodiment, the bivalent linking group has the formula-Q₁-G-Q₂-, wherein

Q₁ and Q₂ are independently absent or selected from substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted alkaryleneor —(CH₂)_(m)—O—(CH₂)_(p)—, wherein

each m and p are, independently, an integer from 1 to about 10;

G is —NH—C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —NH—C(S)—NH—, —NH—O—,NH—C(O)—O—, or —O—CH₂—C(O)—NH—.

Preferred bivalent linking groups are:

In some embodiments, the activated disulfide moiety of the oligomericcompound has the formula —S—S(O)_(n)—R₁, wherein

-   -   n is 0, 1, or 2; and    -   R₁ is selected from substituted or unsubstituted heterocyclic,        substituted or unsubstituted aliphatic, or —C(O)O—R₂, wherein R₂        is substituted or unsubstituted aliphatic.

In further embodiments, the activated disulfide moiety has the formula—S—S(O)₂-substituted or unsubstituted C₁-C₁₂ alkyl or—S—S—C(O)O-substituted or unsubstituted C₁-C₁₂ alkyl. Preferredactivated disulfide moieties are methane thiosulfonate anddithiocarbomethoxy. In further embodiments; the activated disulfide issubstituted or unsubstituted dithiopyridyl, substituted or unsubstituteddithiobenzothiazolyl, or substituted or unsubstituted dithiotetrazolyl.Preferred activated disulfides are 2-dithiopyridyl,2-dithio-3-nitropyridyl, 2-dithio-5-nitropyridyl,2-dithiobenzothiazolyl, N-(C₁-C₁₂ alkyl)-2-dithiopyridyl,2-dithiopyridyl-N-oxide, or 2-dithio-1-methyl-1H-tetrazolyl.

Another aspect of the present invention provides compounds, comprising adouble-stranded oligonucleotide, comprising a first oligonucleotide anda second oligonucleotide, wherein at least one of said first or secondoligonucleotides further comprises a bivalent linking group and anactivated disulfide moiety.

In one embodiment, the composition comprises a double-strandedoligonucleotide, comprising a first oligonucleotide and a secondoligonucleotide, which form a complementary pair of siRNAoligonucleotides. In another embodiment, the composition comprises adouble stranded oligonucleotide, comprising a first oligonucleotide anda second oligonucleotide which form an antisense/sense pair ofoligonucleotides.

In some embodiments, the first or second oligonucleotide of thedouble-stranded oligonucleotide comprises between 8 and 80 nucleobases.In other embodiments, the first or second oligonucleotide of thedouble-stranded oligonucleotide comprises between 10 and 50 nucleobases.In further embodiments, the first or second oligonucleotide of thedouble-stranded oligonucleotide comprises between 15 and 40 nucleobases.In a preferred embodiment, the first or second oligonucleotide of thedouble-stranded oligonucleotide comprises between 15 and 25 nucleobases.

In some embodiments, the bivalent linking group of the double strandedoligonucleotide is a bivalent substituted or unsubstituted aliphaticgroup. In another embodiment, the bivalent linking group has the formula-Q,-G-Q₂-, wherein

Q₁ and Q₂ are independently absent or selected from substituted orunsubstituted C₁-C₁₂ alkylene, substituted or unsubstituted alkaryleneor —(CH₂)_(m)—O—(CH₂)_(p)—, wherein

each m and p are, independently, an integer from 1 to about 10;

G is —NH—C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —NH—C(S)—NH—, —NH—O—,NH—C(O)—O—, or —O—CH₂—C(O)—NH—.

Preferred bivalent linking group s are:

In some embodiments, the activated disulfide moiety of the doublestranded oligonucleotide has the formula —S—S(O)_(n)—R₁, wherein

-   -   n is 0, 1, or 2; and    -   R₁ is selected from substituted or unsubstituted heterocyclic,        substituted or unsubstituted aliphatic, or —C(O)O—R₂, wherein R₂        is substituted or unsubstituted aliphatic.

In another embodiment, the activated disulfide moiety has the formula—S—S(O)₂-substituted or unsubstituted C₁-C₁₂ alkyl or—S—S—C(O)O-substituted or unsubstituted C₁-C₁₂ alkyl. Preferredactivated disulfide moieties are methane thiosulfonate anddithiocarbomethoxy. In further embodiments, the activated disulfide issubstituted or unsubstituted dithiopyridyl, substituted or unsubstituteddithiobenzothiazolyl, or substituted or unsubstituted dithiotetrazolyl.Preferred activated disulfides are 2-dithiopyridyl,2-dithio-3nitropyridyl, 2-dithio-5-nitropyridyl, 2-dithiobenzothiazolyl,N-(C₁-C₁₂ alkyl)-2-dithiopyridyl, 2-dithiopyridyl-N-oxide, or2-dithio-1-methyl-1H-tetrazolyl.

Also within the scope of the present invention are processes of makingany compound described herein via those methods delineated herein. Italso should be understood that the embodiments specifically describedherein are not limiting and it shall be apparent to one of ordinaryskill in the art that additional embodiments not specifically mentionedare within the scope of the instant application.

BRIEF DISCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the covalent attachment ofoligomeric compounds containing activated disulfides to plasma proteinsvia biologically cleavable disulfide linker using a thiol-activatedcysteine.

FIG. 2 2 a) is graphical analysis of the results of the plasma proteinbinding experiment for ISIS350226 in the absence (blue trace) and in thepresence (green trace) of added glutathione; 2 b) depicts release ofISIS350226 from the plasma proteins was facilitated by the addition of 5mM glutathione (a concentration similar to that inside a cell) asdescribed herein in Example 7; 2 c) depicts a similar experiment wascarried out where the release of the PNA-peptide conjugate using 5 mMGSH was monitored over time as shown in FIG. 2 c. depicts the kineticsof plasma protein binding for

FIG. 3 depicts the effect of plasma protein binding on the enzymaticstability of PNA-peptide conjugates without (ISIS309145) and with(ISIS347349) activated disulfide groups.

FIG. 4 depicts tissue distribution in percent recovered dose ofradiolabeled Isis 358346 in male Balb/c mice administered IV either free(a) or pre-bound (b) to plasma.

FIG. 5 depicts the plasma protein binding capacity for ISIS 386773 andISIS 364615 as a function of their plasma concentration.

FIG. 6 depicts the release kinetics of ISIS 386773 from plasma proteinsafter the addition of GSH.

FIG. 7 depicts the binding kinetics of ISIS 386773 to plasma proteins.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.All terms appearing herein which are not specifically defined, shall beaccorded the meaning that one of ordinary skill in the relevant artwould attached to said term.

General Chemistry

The term “alkyl,” as used herein, refers to saturated, straight chain orbranched hydrocarbon moieties containing up to twenty four carbon atoms.The terms “C₁-C₆ alkyl” and “C₁-C₁₂ alkyl” as used herein, refer tosaturated, straight chain or branched hydrocarbon moieties containingone to six carbon atoms and one to twelve carbon atoms respectively.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.

An “aliphatic group,” as used herein, refers to an acyclic, non-aromaticmoiety that may contain any combination of carbon atoms, hydrogen atoms,halogen atoms, oxygen, nitrogen, sulfur, phosphorus or other atoms, andoptionally contain one or more units of unsaturation, e.g., doubleand/or triple bonds. An aliphatic group may be straight chained, orbranched and preferably contains between about 1 and about 24 carbonatoms, more typically between about 1 and about 12 carbon atoms. Inaddition to aliphatic hydrocarbon groups, aliphatic groups include, forexample, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, andpolyimines, for example. Such aliphatic groups may be furthersubstituted.

Suitable substituents of the present invention include, but are notlimited to, —F, —Cl, —Br, —I, —OH, protected hydroxy, aliphatic ethers,aromatic ethers, oxo, azido, imino, oximino, —NO₂, —CN, —COOH,—C₁-C₁₂-alkyl optionally substituted, C₂-C₁₂-alkenyl optionallysubstituted, —C₂-C₁₂-alkynyl optionally substituted, —NH₂, protectedamino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkynyl,—NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl,-dialkylamino, -diarylamino, -diheteroarylamino, —O—C₁-C₁₂-alkyl,—O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkynyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl,—O-heteroaryl, —O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl,—C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkynyl, —C(O)—C₃-C₁₂-cycloalkyl,—C(O)-aryl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂,—CONH—C₁-C₁₂-alkyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkynyl,—CONH—C₃-C₁₂-cycloalkyl, —CONH-aryl, —CONH-heteroaryl,—CONH-heterocycloalkyl, —CO₂-C₁-C₁₂-alkyl, —CO₂—C₂-C₁₂-alkenyl,—CO₂—C₂-C₁₂-alkynyl, —CO₂—C₃-C₁₂-cycloalkyl, —CO₂-aryl, —CO₂-heteroaryl,—CO₂-heterocycloalkyl, —OCO₂H, —OCO₂—C₂-C₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl,—OCO₂—C₂-C₁₂-alkynyl, —OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl,—OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkynyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl,—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkynyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)—heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkynyl, —NHC(O)NH—C₃-C₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH₂, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkynyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkynyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl, —SO₂NH₂, —SO₂NH—C₁-C₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, aryl, arylalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, methoxymethoxy, methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl. It is understood that the aryls, heteroaryls, alkylsand the like can be further substituted.

The term “alkenyl” as used herein, refers to a straight chain orbranched hydrocarbon moiety containing up to twenty four carbon atomshaving at least one carbon-carbon double bond. The terms “C₂-C₆ alkenyl”and “C₂-C₁₂ alkenyl,” as used herein refer to straight chain or branchedhydrocarbon moieties containing two to six carbon atoms and two totwelve carbon atoms respectively and having at least one carbon-carbondouble bond. Examples of alkenyl groups include, but are not limited to,ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, alkadienes and thelike.

The term “substituted alkenyl,” as used herein, refers to an “alkenyl”or “C₂-C₁₂ alkenyl” or “C₂-C₆ alkenyl” group as previously defined,substituted by one, two, three or more substituents.

The term “alkynyl” as used herein, refers to a straight chain orbranched hydrocarbon moiety containing up to twenty four carbon atomsand having at least one carbon-carbon triple bond. The terms “C₂-C₆alkynyl” and “C₂-C₁₂ alkynyl,” as used herein, refer to straight chainor branched hydrocarbon moieties containing two to six carbon atoms andtwo to twelve carbon atoms respectively and having at least onecarbon-carbon triple bond. Examples of alkynyl groups include, but arenot limited to, ethynyl, 1-propynyl, 1-butynyl, and the like.

The term “substituted alkynyl,” as used herein, refers to an “alkynyl”or “C₂-C₆ alkynyl” or “C₂-C₁₂ alkynyl” group as previously defined,substituted by one, two, three or more substituents.

The term “alkoxy,” as used herein, refers to an aliphatic group, aspreviously defined, attached to the parent molecular moiety through anoxygen atom. Examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,n-pentoxy, neopentoxy, n-hexoxy and the like.

The term “substituted alkoxy,” as used herein, refers to an alkoxy groupas previously defined substituted with one, two, three or moresubstituents.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “aryl” or “aromatic,” as used herein, refer to a mono-, bi- ortri-cyclic carbocyclic ring system having one, two or three aromaticrings. Examples of aryl groups include, but not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.

The terms “substituted aryl” or “substituted aromatic,” as used herein,refer to an aryl or aromatic group as previously defined substituted byone, two, three or more substituents.

The term “arylalkyl,” as used herein, refers to an aryl group attachedto the parent molecular moiety via a C₁-C₃ alkyl or C₁-C₆ alkyl residue.Examples include, but are not limited to, benzyl, phenethyl and thelike.

The term “substituted arylalkyl,” as used herein, refers to an arylalkylgroup as previously defined, substituted by one, two, three or moresubstituents.

The terms “heteroaryl” or “heteroaromatic,” as used herein, refer to amono-, bi-, or tri-cyclic aromatic radical or ring having from five toten ring atoms of which at least one ring atom is selected from S, O andN; zero, one, two or three ring atoms are additional heteroatomsindependently selected from S, O and N; and the remaining ring atoms arecarbon, wherein any N or S contained within the ring may be optionallyoxidized. Examples of heteroaryl groups include, but are not limited to,pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,quinoxalinyl, and the like. The heteroaromatic ring may be bonded to theparent molecular moiety through a carbon or hetero atom.

The terms “substituted heteroaryl” or “substituted heteroaromatic,” asused herein, refer to a heteroaryl or heteroaromatic group as previouslydefined, substituted by one, two, three, or more substituents.

The term “alicyclic,” as used herein, denotes a monovalent group derivedfrom a monocyclic or bicyclic saturated carbocyclic ring compound by theremoval of a single hydrogen atom. Examples include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, bicyclo [2.2.2] octyl and the like.

The term “substituted alicyclic,” as used herein, refers to an alicyclicgroup as previously defined, substituted by one, two, three or moresubstituents.

The terms “heterocyclic,” or “heterocycloalkyl” as used herein, refer toa non-aromatic ring, comprising three or more ring atoms, or a bi- ortri-cyclic fused system, where (i) each ring contains between one andthree heteroatoms independently selected from oxygen, sulfur andnitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfurheteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom mayoptionally be quaternized, (iv) any of the above rings may be fused to abenzene ring, and (v) the remaining ring atoms are carbon atoms whichmay be optionally oxo-substituted. Examples of heterocyclic groupsinclude, but are not limited to, [1,3]dioxolane, pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and thelike.

The term “substituted heterocyclic,” as used herein, refers to aheterocyclic group, as previously defined, substituted by one, two,three or more substituents.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined, attached to the parent molecular moiety via analkyl residue. Examples include, but are not limited to,pyridinylmethyl, pyrimidinylethyl and the like.

The term “substituted heteroarylalkyl,” as used herein, refers to aheteroarylalkyl group, as previously defined, substituted by one, two,three or more substituents.

The term “alkylamino” refers to a group having the structure —NH-alkyl.

The term “dialkylamino” refers to a group having the structure—N(alkyl)₂ and cyclic amines. Examples of dialkylamino include, but arenot limited to, dimethylamino, diethylamino, methylethylamino,piperidino, morpholino and the like.

The term “alkoxycarbonyl” refers to an ester group, i.e., an alkoxygroup, attached to the parent molecular moiety through a carbonyl groupsuch as methoxycarbonyl, ethoxycarbonyl, and the like.

The term “carboxaldehyde,” as used herein, refers to a group of formula—CHO.

The term “carboxy,” as used herein, refers to a group of formula —COOH.

The term “carboxamide,” as used herein, refers to a group of formula—C(O)NH-alkyl or —C(O)N(alkyl)₂, —C(O)NH₂, —NHC(O)alkyl,—N(alkyl)C(O)(alkyl) and the like.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect a hydroxyl, amino or thiolgroup against undesired reactions during synthetic procedures. Aftersaid synthetic procedure(s) the protecting group as described herein maybe selectively removed. Protecting groups as known in the art aredescribed generally in T. H. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of hydroxyl protecting groups include, but are not limited to,benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl (BOC),isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl (Alloc), acetyl (Ac), formyl,chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl(Bz), methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilyl ethyl,1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl (Bn),para-methoxybenzyldiphenylmethyl, triphenylmethyl (trityl),4,4′-dimethoxytriphenylmethyl (DMT), substituted or unsubstituted9-(9-phenyl)xanthenyl (pixyl), tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxyl protecting groups for the present invention are DMTand substituted or unsubstituted pixyl.

Amino protecting groups as known in the art are described generally inT. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,3rd edition, John Wiley & Sons, New York (1999). Examples of aminoprotecting groups include, but are not limited to, t-butoxycarbonyl(BOC), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl, and thelike.

Thiol protecting groups as known in the art are described generally inT. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,3rd edition, John Wiley & Sons, New York (1999). Examples of thiolprotecting groups include, but are not limited to, triphenylmethyl(Trt), benzyl (Bn), and the like.

The term “protected hydroxyl group,” as used herein, refers to ahydroxyl group protected with a protecting group, as previously defined.

The term “protected amino group,” as used herein, refers to an aminogroup protected with a protecting group, as previously defined.

The term “protected thiol group,” as used herein, refers to a thiolgroup protected with a protecting group, as previously defined.

The term “acyl” includes residues derived from substituted orunsubstituted acids including, but not limited to, carboxylic acids,carbamic acids, carbonic acids, sulfonic acids, and phosphorous acids.Examples include aliphatic carbonyls, aromatic carbonyls, aliphaticsulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphatesand aliphatic phosphates.

The term “aprotic solvent,” as used herein, refers to a solvent that isrelatively inert to proton activity, i.e., not acting as a proton-donor.Examples include, but are not limited to, hydrocarbons, such as hexane,toluene and the like, halogenated hydrocarbons, such as methylenechloride, ethylene chloride, chloroform, and the like, heterocycliccompounds, such as tetrahydrofuran, N-methylpyrrolidinone and the like,and ethers such as diethyl ether, bis-methoxymethyl ether and the like.Such compounds are well known to those skilled in the art, and it willbe obvious to those skilled in the art that individual solvents ormixtures thereof may be preferred for specific compounds and reactionconditions, depending upon such factors as the solubility of reagents,reactivity of reagents and preferred temperature ranges, for example.Further discussions of aprotic solvents may be found in organicchemistry textbooks or in specialized monographs, for example: OrganicSolvents Physical Properties and Methods of Purification, 4th ed.,edited by John A. Riddick et al, Vol. II, in the Techniques of ChemistrySeries, John Wiley & Sons, NY, 1986. Aprotic solvents useful in theprocesses of the present invention include, but are not limited to,toluene, acetonitrile, DMF, THF, dioxane, MTBE, diethylether, NMP,acetone, hydrocarbons, and haloaliphatics.

The term “protic solvent” or “protogenic solvent” as used herein, refersto a solvent that tends to provide protons, such as an alcohol, forexample, methanol, ethanol, propanol, isopropanol, butanol, t-butanol,and the like. Such solvents are well known to those skilled in the art,and it will be obvious to those skilled in the art that individualsolvents or mixtures thereof may be preferred for specific compounds andreaction conditions, depending upon such factors as the solubility ofreagents, reactivity of reagents and preferred temperature ranges, forexample. Further discussions of protic solvents may be found in organicchemistry textbooks or in specialized monographs, for example: OrganicSolvents Physical Properties and Methods of Purification, 4th ed.,edited by John A. Riddick et al., Vol. II, in the Techniques ofChemistry Series, John Wiley & Sons, NY, 1986.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. Further methods ofsynthesizing the compounds of the formulae herein will be evident tothose of ordinary skill in the art. Additionally, the various syntheticsteps may be performed in an alternate sequence or order to give thedesired compounds. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to designate a particularconfiguration unless the text so states; thus a carbon-carbon doublebond or carbon-heteroatom double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion.

Gene Modulation

The term “subject” as used herein refers to an animal. Preferably theanimal is a mammal. More preferably the mammal is a human. A subjectalso refers to, for example, monkeys, dogs, cats, horses, cows, pigs,rabbits, rats, mice, guinea pigs, and the like.

As used herein, the term “target nucleic acid” or “nucleic acid target”is used for convenience to encompass any nucleic acid capable of beingtargeted including without limitation DNA, RNA (including pre-mRNA andmRNA or portions thereof) transcribed from such DNA, and also cDNAderived from such RNA. In one embodiment of the invention, the targetnucleic acid is a messenger RNA. In another embodiment, the degradationof the targeted messenger RNA is facilitated by a RISC complex that isformed with oligomeric compounds of the invention. In anotherembodiment, the degradation of the targeted messenger RNA is facilitatedby a nuclease such as RNaseH.

The hybridization of an oligomeric compound of this invention with itstarget nucleic acid is generally referred to as “antisense”.Consequently, one mechanism in the practice of some embodiments of theinvention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently suitable to target specificnucleic acid molecules and their functions for such antisenseinhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA.

In the context of the present invention, “modulation” and “modulation ofexpression” mean either an increase (stimulation) or a decrease(inhibition) in the amount or levels of a nucleic acid molecule encodingthe gene, e.g., DNA or RNA. Inhibition is often the desired form ofmodulation of expression and mRNA is often a desired target nucleicacid.

The compounds and methods of the present invention are also useful inthe study, characterization, validation and modulation of smallnon-coding RNAs. These include, but are not limited to, microRNAs(miRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA),small temporal RNAs (stRNA) and tiny non-coding RNAs (tncRNA) or theirprecursors or processed transcripts or their association with othercellular components.

Small non-coding RNAs have been shown to function in variousdevelopmental and regulatory pathways in a wide range of organisms,including plants, nematodes and mammals. MicroRNAs are small non-codingRNAs that are processed from larger precursors by enzymatic cleavage andinhibit translation of mRNAs. stRNAs, while processed from precursorsmuch like miRNAs, have been shown to be involved in developmental timingregulation. Other non-coding small RNAs are involved in events asdiverse as cellular splicing of transcripts, translation, transport, andchromosome organization.

As modulators of small non-coding RNA function, the compounds of thepresent invention find utility in the control and manipulation ofcellular functions or processes such as regulation of splicing,chromosome packaging or methylation, control of developmental timingevents, increase or decrease of target RNA expression levels dependingon the timing of delivery into the specific biological pathway andtranslational or transcriptional control. In addition, the compounds ofthe present invention can be modified in order to optimize their effectsin certain cellular compartments, such as the cytoplasm, nucleus,nucleolus or mitochondria.

The compounds of the present invention can further be used to identifycomponents of regulatory pathways of RNA processing or metabolism aswell as in screening assays or devices.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound”refers to a polymeric structure capable of hybridizing a region of anucleic acid molecule. This term includes oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics andcombinations of these. Oligomeric compounds are routinely preparedlinearly but can be joined or otherwise prepared to be circular and mayalso include branching. Oligomeric compounds can include double strandedconstructs such as for example two strands hybridized to form doublestranded compounds. The double stranded compounds can be linked orseparate and can include overhangs on the ends. In general, anoligomeric compound comprises a backbone of linked momeric subunitswhere each linked momeric subunit is directly or indirectly attached toa heterocyclic base moiety. Oligomeric compounds may also includemonomeric subunits that are not linked to a heterocyclic base moietythereby providing abasic sites. The linkages joining the monomericsubunits, the sugar moieties or surrogates and the heterocyclic basemoieties can be independently modified giving rise to a plurality ofmotifs for the resulting oligomeric compounds including hemimers,gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond, however, open linear structures aregenerally desired. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside linkagesof the oligonucleotide. The normal internucleoside linkage of RNA andDNA is a 3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA). This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring portions which function ina similar manner to oligonulceotides. Such non-naturally occurringoligonucleotides are often desired over the naturally occurring formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers to asequence of nucleosides that are joined by internucleoside linkages thatdo not have phosphorus atoms. Internucleoside linkages of this typeinclude short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixedheteroatom cycloalkyl, one or more short chain heteroatomic and one ormore short chain heterocyclic. These internucleoside linkages include,but are not limited to, siloxane, sulfide, sulfoxide, sulfone, acetyl,formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl,alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate,sulfonamide, amide and others having mixed N, O, S and CH₂ componentparts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Further included in the present invention are oligomeric compounds suchas antisense oligomeric compounds, antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, alternatesplicers, primers, probes, and other oligomeric compounds whichhybridize to at least a portion of the target nucleic acid. As such,these oligomeric compounds may be introduced in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense oligomeric compoundswhich are “DNA-like” elicit RNAse H. Activation of RNase H, therefore,results in cleavage of the RNA target, thereby greatly enhancing theefficiency of oligonucleotide-mediated inhibition of gene expression.Similar roles have been postulated for other ribonucleases such as thosein the RNase III and ribonuclease L family of enzymes.

While one form of antisense oligomeric compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

In addition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodification so long as these other modifications either alone or incombination with other nucleosides enhance one or more of the desiredproperties described above. Thus, for nucleotides that are incorporatedinto oligonucleotides of the invention, these nucleotides can have sugarportions that correspond to naturally-occurring sugars or modifiedsugars. Representative modified sugars include carbocyclic or acyclicsugars, sugars having substituent groups at one or more of their 2′, 3′or 4′ positions and sugars having substituents in place of one or morehydrogen atoms of the sugar. Additional nucleosides amenable to thepresent invention having altered base moieties and or altered sugarmoieties are disclosed in U.S. Pat. No. 3,687,808 and PCT applicationPCT/US89/02323.

The term “nucleobase,” as used herein, is intended to by synonymous with“nucleic acid base or mimetic thereof.” In general, a nucleobase is anysubstructure that contains one or more atoms or groups of atoms capableof hydrogen bonding to a base of an oligonucleotide.

As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

Modified Nucleobases

Modified nucleobases include, but are not limited to, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Certain of these nucleobases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

The term “universal base” as used herein, refers to a moiety that may besubstituted for any base. The universal base need not contribute tohybridization, but should not significantly detract from hybridizationand typically refers to a monomer in a first sequence that can pair witha naturally occuring base, i.e A, C, G, T or U at a correspondingposition in a second sequence of a duplex in which one or more of thefollowing is true: (1) there is essentially no pairing between the two;or (2) the pairing between them occurs non-discriminantly with each ofthe naturally occurring bases and without significant destabilization ofthe duplex. Exemplary universal bases include, without limitation,inosine, 5-nitroindole and 4-nitrobenzimidazole.

Additional examples of universal bases include, but are not limited to,those shown below. For further examples and descriptions of universalbases see Survey and summary: the applications of universal DNA baseanalogs. Loakes, D. Nucleic Acids Research, 2001, 29, 12, 2437-2447.

The term “hydrophobic base” as used herein, refers to a monomer in afirst sequence that can pair with a naturally occuring base, i.e A, C,G, T or U at a corresponding position in a second sequence of a duplexin which one or more of the following is true: (1) the hydrophobic baseacts as a non-polar close size and shape mimic (isostere) of one of thenaturally occurring nucleosides; or (2) the hydrophobic base lacks allhydrogen bonding functionality on the Watson-Crick pairing edge.

Examples of adenine isosteres include, but are not limited to thoseshown below. For further examples and definitions of adenine isosteressee Probing the requirements for recognition and catalysis in Fpg andMutY with nonpolar adenine isosteres. Francis, A W, Helquist, S A, Kool,E T, David, S S. J. Am. Chem. Soc., 2003, 125, 16235-16242 or Structureand base pairing properties of a replicable nonpolar isostere fordeoxyadenosine. Guckian, K M, Morales, J C, Kool, E T. J. Org. Chem.,1998, 63, 9652-96565.

A non-limiting example of a cytosine isostere is2-fluoro-4-methylbenzene deoxyribonucleoside, shown below. Foradditional information on cytosine isosteres see Hydrolysis of RNA/DNAhybrids containing nonpolar pyrimidine isosteres defines regionsessential for HIV type polypurine tract selection. Rausch, J W, Qu, J,Yi-Brunozzi H Y, Kool, E T, LeGrice, S F J. Proc. Natl. Acad. Sci.,2003, 100, 11279-11284.

A non-limiting example of a guanosine isostere is4-fluoro-6-methylbenzimidazole deoxyribonucleoside, shown below. Foradditional information on guanosine isosteres, see A highly effectivenonpolar isostere of doeoxguanosine: synthesis, structure, stacking andbase pairing. O'Neil, B M, Ratto, J E, Good, K L, Tahmassebi, D C,Helquist, S A, Morales, J C, Kool, E T. J. Org. Chem., 2002, 67,5869-5875.

A non-limiting example of a thymidine isostere is 2,4-difluoro-5-toluenedeoxyribonucleoside, shown below. For additional information onthymidine isosteres see A thymidine triphosphate shape analog lackingWatson-Crick pairing ability is replicated with high sequenceselectivity. Moran, S, Ren, RX-F, Kool, E T. Proc. Natl. Acad. Sci.,1997, 94, 10506-10511 or Difluorotoluene, a nonpolar isostere forthymidine, codes specifically and efficiently for adenine in DNAreplication. J. Am. Chem. Soc. 1997, 119, 2056-2057.

The term “promiscuous base” as used herein, refers to a monomer in afirst sequence that can pair with a naturally occuring base, i.e A, C,G, T or U at a corresponding position in a second sequence of a duplexin which the promiscuous base can pair non-discriminantly with more thanone of the naturally occurring bases, i.e. A, C, G, T, U. Non-limitingexamples of promiscuous bases are6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one andN⁶-methoxy-2,6-diaminopurine, shown below. For further information, seePolymerase recognition of synthetic oligodeoxyribonucleotidesincorporating degenerate pyrimidine and purine bases. Hill, F.; Loakes,D.; Brown, D. M. Proc. Natl. Acad. Sci., 1998, 95, 4258-4263.

The term “size expanded base” as used herein, refers to analogs ofnaturally occurring nucleobases that are larger in size and retain theirWatson-Crick pairing ability. Tow non-limiting examples of size-expandedbases are shown below. For further discussions of size expanded basessee A four-base paired genetic helix with expanded size. Liu, B, Gao, J,Lynch, S R, Saito, D, Maynard, L, Kool, E T., Science, 2003, 302,868-871 and Toward a new genetic system with expanded dimension: sizeexpanded analogues of deoxyadenosine and thymidine. Liu, H, Goa, J,Maynard, Y, Saito, D, Kool, E T, J. Am. Chem. Soc. 2004, 126, 1102-1109and Expanded-Size Bases in Naturally Sized DNA: Evaluation of StericEffects in Watson-Crick Pairing. Gao, J, Liu, H, Kool, E, J. Am. Chem.Soc. 2004, 126, 11826-11831.

The term “fluorinated nucleobase” as used herein, refers to a nucleobaseor nucleobase analog, wherein one or more of the aromatic ringsubstituents is a fluoroine atom. It may be possible that all of thering substituents are fluoroine atoms. Some non-limiting examples offluorinated nucleobase are shown below. For further examples offluorinated nucleobases see fluorinated DNA bases as probes ofelectrostatic effects in DNA base stacking. Lai, J S, Q U, J, Kool, E T,Angew. Chem. Int. Ed., 2003, 42, 5973-5977 and Selective pairing ofpolyfluorinated DNA bases, Lai, J S, Kool, E T, J. Am. Chem. Soc., 2004,126, 3040-3041 and The effect of universal fluorinated nucleobases onthe catalytic activity of ribozymes, Kloppfer, A E, Engels, J W,Nucleosides, Nucleotides & Nucleic Acids, 2003, 22, 1347-1350 andSynthesis of 2′aminoalkyl-substituted fluorinated nucleobases and theirinfluence on the kinetic properties of hammerhead ribozymes, Klopffer, AE, Engels, J W, Chem Bio Chem., 2003, 5, 707-716.

In some embodiments of the invention, oligomeric compounds, e.g.oligonucleotides, are prepared having polycyclic heterocyclic compoundsin place of one or more heterocyclic base moieties. A number oftricyclic heterocyclic compounds have been previously reported. Thesecompounds are routinely used in antisense applications to increase thebinding properties of the modified strand to a target strand. The moststudied modifications are targeted to guanosines hence they have beentermed G-clamps or cytidine analogs. Many of these polycyclicheterocyclic compounds have the general formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second oligonucleotide include 1,3-diazaphenoxazine-2-one(R₁₀═O, R₁₁-R₁₄═H) [Kurchavov, et al., Nucleosides and Nucleotides,1997, 16, 1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁-R₁₄═H),[Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀═O, R₁₁═ —O—(CH₂)₂—NH₂,R₁₂₋₁₄═H) [Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120,8531-8532]. Binding studies demonstrated that a single incorporationcould enhance the binding affinity of a model oligonucleotide to itscomplementary target DNA or RNA with a ΔT_(m) of up to 18° relative to5-methyl cytosine (dC5^(me)), which is the highest known affinityenhancement for a single modification, yet. On the other hand, the gainin helical stability does not compromise the specificity of theoligonucleotides. The T_(m) data indicate an even greater discriminationbetween the perfect match and mismatched sequences compared to dC5^(me).It was suggested that the tethered amino group serves as an additionalhydrogen bond donor to interact with the Hoogsteen face, namely the O6,of a complementary guanine thereby forming 4 hydrogen bonds. This meansthat the increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992,which issued on Dec. 28, 1999, the contents of both are commonlyassigned with this application and are incorporated herein in theirentirety. Such compounds include those having the formula:

Wherein R₁₁ includes (CH₃)₂N—(CH₂)₂—O—; H₂N—(CH₂)₃—;Ph-CH₂—O—C(═O)—N(H)—(CH₂)₃—; H₂N—; Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—;Phthalimidyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—; Ph-CH₂—O—C(═O)—N(H)—(CH₂)₂—O—;Ph-CH₂—O—C(═O)—N(H)—(CH₂)₃—O—; (CH₃)₂N—N(H)—(CH₂)₂—O—;Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₂—O—;Fluorenyl-CH₂—O—C(═O)—N(H)—(CH₂)₃—O—; H₂N—(CH₂)₂—O—CH₂—;N₃—(CH₂)₂—O—CH₂—; H₂N—(CH₂)₂—O—, and NH₂C(═NH)NH—.

Also disclosed are tricyclic heterocyclic compounds of the formula:

wherein:

R_(10a) is O, S or N—CH₃; R_(11a) is A(Z)_(x1), wherein A is a spacerand Z independently is a label bonding group optionally bonded to adetectable label, but R_(11a) is not amine, protected amine, nitro orcyano; and R_(b) is independently —CH═, —N═, —C(C₁₋₈ alkyl)=or—C(halogen)=, but no adjacent R_(b) are both —N═, or two adjacent R_(b)are taken together to form a ring having the structure:

where R_(c) is independently —CH═, —N═, —C(C₁₋₈ alkyl)= or —C(halogen)=,but no adjacent R_(b) are both —N═.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity [Lin, K.-Y.; Matteucci, M.J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement waseven more pronounced in case of G-clamp, as a single substitution wasshown to significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further tricyclic and tetracyclic heteroaryl compounds amenable to thepresent invention include those having the formulas:

wherein R₁₄ is NO₂ or both R₁₄ and R₁₂ are independently —CH₃. Thesynthesis of these compounds is dicslosed in U.S. Pat. No. 5,434,257,which issued on Jul. 18, 1995, U.S. Pat. No. 5,502,177, which issued onMar. 26, 1996, and U.S. Pat. No. 5,646,269, which issued on Jul. 8,1997, the contents of which are commonly assigned with this applicationand are incorporated herein in their entirety (hereinafter refered to asthe “'257, '177 and '269 patents”).

Further tricyclic heterocyclic compounds amenable to the presentinvention also disclosed in the '257, '177 and '269 patents includethose having the formula:

wherein a and b are independently 0 or 1 with the total of a and b being0 or 1; A is N, C or CH; Y is S, O, C═O, NH or NCH₂, R⁶; Z is C═O; B istaken together with A to form an aryl or heteroaryl ring structurecomprising 5 or 6 ring atoms wherein the heteroaryl ring comprises asingle O ring heteroatom, a single N ring heteroatom, a single S ringheteroatom, a single O and a single N ring heteroatom separated by acarbon atom, a single S and a single N ring heteroatom separated by a Catom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ringheteroatoms at least 2 of which are separated by a carbon atom, andwherein the aryl or heteroaryl ring carbon atoms are unsubstituted withother than H or at least 1 nonbridging ring carbon atom is fubstitutedwith R²⁰ or ═O; or Z is taken together with A to form an aryl ringstructure comprising 6 ring atoms wherein the aryl ring carbon atoms areunsubstituted with other than H or at least 1 nonbridging ring carbonatom is substituted with R⁶ or ═O; R⁶ is independently H, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, NO₂, N(R³)₂, CN or halo, or an R⁶ is takentogether with an adjacent B group R⁶ to complete a phenyl ring; R²⁰ is,independently, H, C₁₋₆ alkyl, C₂₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,NO₂, N(R²¹)₂, CN, or halo, or an R²⁰ is taken together with an adjacentR²⁰ to complete a ring containing 5 or 6 ring atoms, and tautomers,solvates and salts thereof; R²¹ is, independently, H or a protectinggroup; R³ is a protecting group or H; and tautomers, solvates and saltsthereof.

More specific examples of bases included in the “257, 177 and 269”Patents are compounds of the formula:

wherein each R₁₆, is, independently, selected from hydrogen and varioussubstituent groups. Further polycyclic base moieties having the formula:

wherein: A₆ is O or S; A₇ is CH₂, N—CH₃, O or S; each A₈ and A₉ ishydrogen or one of A₈ and A₉ is hydrogen and the other of A₈ and A₉ isselected from the group consisting of:

wherein: G is —CN, —OA₁₀, —SA₁₀, —N(H)A₁₀, —ON(H)A₁₀ or —C(═NH)N(H)A₁₀;Q₁ is H, —NHA₁₀, —C(═O)N(H)A₁₀, —C(═S)N(H)A₁₀ or —C(═NH)N(H)A₁₀; each Q₂is, independently, H or Pg; A₁₀ is H, Pg, substituted or unsubstitutedC₁-C₁₀ alkyl, acetyl, benzyl, —(CH₂)_(p3)NH₂, —(CH₂)_(p3)N(H)Pg, a D orL α-amino acid, or a peptide derived from D, L or racemic α-amino acids;Pg is a nitrogen, oxygen or thiol protecting group; each p1 is,independently, from 2 to about 6; p2 is from 1 to about 3; and p3 isfrom 1 to about 4; are disclosed in U.S. patent application Ser. No.09/996,292 filed Nov. 28, 2001, which is commonly owned with the instantapplication, and is herein incorporated by reference.

Some particularly useful oligomeric compounds of the invention containat least one nucleoside having one, two, three, or more aliphaticsubstituents, an RNA cleaving group, a reporter group, an intercalator,a group for improving the pharmacokinetic properties of an oligomericcompound, or a group for improving the pharmacodynamic properties of anoligomeric compound, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferredmodification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE. Representative aminooxy substituentgroups are described in co-owned U.S. patent application Ser. No.09/344,260, filed Jun. 25, 1999, entitled “Aminooxy-FunctionalizedOligomers”; and U.S. patent application Ser. No. 09/370,541, filed Aug.9, 1999, entitled “Aminooxy-Functionalized Oligomers and Methods forMaking Same;” hereby incorporated by reference in their entirety.

Other particularly advantageous 2′-modifications include 2′-methoxy(2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F).Similar modifications may also be made at other positions on nucleosidesand oligomers, particularly the 3′ position of the sugar on the 3′terminal nucleoside or at a 3′-position of a nucleoside that has alinkage from the 2′-position such as a 2′-5′ linked oligomer and at the5′ position of a 5′ terminal nucleoside. Oligomers may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/468,037, filed onJun. 5, 1995, also herein incorporated by reference.

Representative guanidino substituent groups that are shown in formulaare disclosed in co-owned U.S. patent application Ser. No. 09/349,040,entitled “Functionalized Oligomers”, filed Jul. 7, 1999, issue fee paidon Oct. 23, 2002.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Modified Oligonucleotides”, filed Aug.6, 1999, hereby incorporated by reference in its entirety. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. Therespective ends of this linear polymeric structure can be joined to forma circular structure by hybridization or by formation of a covalentbond, however, open linear structures are generally preferred. Withinthe oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide. The normal internucleoside linkage of RNA and DNA is a3′ to 5′ phosphodiester linkage.

The oligomeric compounds in accordance with this invention can comprisefrom about 8 to about 80 nucleobases (i.e., from about 8 to about 80linked nucleosides). One of ordinary skill in the art will appreciatethat the invention embodies oligomeric compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 12to 50 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length,or any range therewithin.

Suitable oligomeric compounds are oligonucleotides from about 12 toabout 50 nucleobases or from about 15 to about 30 nucleobases.

Oligomer and Monomer Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally desired. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside linkage or in conjunctionwith the sugar ring the backbone of the oligonucleotide. The normalinternucleoside linkage that makes up the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage.

Chimeric Oligomeric Compounds

It is not necessary for all positions in an oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within anoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative U.S.patents that teach the preparation of such hybrid structures include,but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;5,652,355; 5,652,356; and 5,700,922, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligomer Mimetics

Another group of oligomeric compounds amenable to the present inventionincludes oligonucleotide mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligomeric compounds whereinonly the furanose ring or both the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.

wherein

each Bx is independently a nucleobase;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the ω-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 2 to about 50.

Another modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage can be prefereably a methylene,ethylene (referred to in the art as ENA), or (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 to 10 (Singh etal., Chem. Commun., 1998, 4, 455-456). LNA analogs display very highduplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10C), stability towards 3′-exonucleolytic degradation and good solubilityproperties. The basic structure of LNA showing the bicyclic ring systemis shown below:

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638). The authors have demonstrated that LNAs confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNAs have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Further oligonucleotide mimetics have been prepared to incude bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J.Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogshave been oligomerized using the phosphoramidite approach and theresulting oligomeric compounds containing tricyclic nucleoside analogshave shown increased thermal stabilities (Tm's) when hybridized to DNA,RNA and itself. Oligomeric compounds containing bicyclic nucleosideanalogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in abackbone the backbone. This class of olignucleotide mimetic is reportedto have useful physical and biological and pharmacological properties inthe areas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Modified Internucleoside Linkages

Specific examples of antisense oligomeric compounds useful in thisinvention include oligonucleotides containing modified e.g.non-naturally occurring internucleoside linkages. As defined in thisspecification, oligonucleotides having modified internucleoside linkagesinclude internucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage(phosphorothioate) did not significantly interfere with RNAi activity.Based on this observation, it is suggested that certain oligomericcompounds of the invention can also have one or more modifiedinternucleoside linkages. One phosphorus containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage.

Modified oligonucleotide backbones containing a phosphorus atom thereininclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In some embodiments of the invention, oligomeric compounds have one ormore phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Suitableamide internucleoside linkages are disclosed in the above referencedU.S. Pat. No. 5,602,240.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted sugar moieties. Suitable oligomeric compounds comprise asugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-,S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other oligonucleotides comprise a sugar substituent groupselected from: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Onemodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. Another modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be inthe arabino (up) position or ribo (down) position. Similar modificationsmay also be made at other positions on the oligomeric compound,particularly the 3′ position of the sugar on the 3′ terminal nucleosideor in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide. Oligomeric compounds may also have sugar mimetics such ascyclobutyl moieties in place of the pentofuranosyl sugar. RepresentativeU.S. patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety. Further representative sugar substituentgroups include groups of formula Ia or Ib:

wherein:

R_(b) is O, S or NH;

R_(d) is a single bond, O, S or C(═O);

R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula Ic;

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

R_(r) is —R_(x)—R_(y);

each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_(u) and R_(v), together form a phthalimido moiety withthe nitrogen atom to which they are attached;

each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R_(k) is hydrogen, an amino protecting group or —R_(x)—R_(y);

R_(p) is hydrogen, an amino protecting group or —R_(x)—R_(y);

R_(x) is a bond or a linking moiety;

R_(y) is a chemical functional group, a conjugate group or a solidsupport medium;

each R_(m) and R_(n) is, independently, H, an amino protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein thesubstituent groups are selected from hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl where said acyl isan acid amide or an ester;

or R_(m) and R_(n), together, are an amino protecting group, are joinedin a ring structure that optionally includes an additional heteroatomselected from N and O or are a chemical functional group;

R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

R_(f), R_(g) and R_(h) comprise a ring system having from about 4 toabout 7 carbon atoms or having from about 3 to about 6 carbon atoms and1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m)) OR_(k),halo, SR_(k) or CN;

m_(a) is 1 to about 10;

each mb is, independently, 0 or 1;

mc is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups of Formula Ia are disclosed in U.S.patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled“Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by referencein its entirety.

Representative cyclic substituent groups of Formula Ib are disclosed inU.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998,entitled “RNA Targeted 2′-Oligomeric compounds that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Sugar substituent groups also include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)H)]₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups that are shown in formula Icare disclosed in co-owned U.S. patent application Ser. No. 09/349,040,entitled “Functionalized Oligomers”, filed Jul. 7, 1999, herebyincorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Conjugates

Another substitution that can be appended to the oligomeric compounds ofthe invention involves the linkage of one or more moieties or conjugateswhich enhance the activity, cellular distribution or cellular uptake ofthe resulting oligomeric compounds. In one embodiment such modifiedoligomeric compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, poly-ethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Representative conjugate groups are disclosedin International Patent Application PCT/US92/09196, filed Oct. 23, 1992the entire disclosure of which is incorporated herein by reference.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

3′-Endo Modifications

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as illustrated in Scheme 1a, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation. Along similar lines, oligomeric triggers of RNAi responsemight be composed of one or more nucleosides modified in such a way thatconformation is locked into a C3′-endo type conformation, i.e. LockedNucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), andethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &Medicinal Chemistry Letters (2002), 12, 73-76.) Representative examplesof modified nucleosides amenable to the present invention include, butare not limited to those shown below:

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligoncleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below.) Nucleosides knownto be inhibitors/substrates for RNA dependent RNA polymerases (forexample HCV NS5B

In one aspect, the present invention is directed to oligonucleotidesthat are prepared having enhanced properties compared to native RNAagainst nucleic acid targets. A target is identified and anoligonucleotide is selected having an effective length and sequence thatis complementary to a portion of the target sequence. Each nucleoside ofthe selected sequence is scrutinized for possible enhancingmodifications. A preferred modification would be the replacement of oneor more RNA nucleosides with nucleosides that have the same 3′-endoconformational geometry. Such modifications can enhance chemical andnuclease stability relative to native RNA while at the same time beingmuch cheaper and easier to synthesize and/or incorporate into anoligonulceotide. The selected sequence can be further divided intoregions and the nucleosides of each region evaluated for enhancingmodifications that can be the result of a chimeric configuration.Consideration is also given to the 5′ and 3′-termini as there are oftenadvantageous modifications that can be made to one or more of theterminal nucleosides. The oligomeric compounds of the present inventioninclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double stranded sequence or sequences.Further modifications are also considered such as internucleosidelinkages, conjugate groups, substitute sugars or bases, substitution ofone or more nucleosides with nucleoside mimetics and any othermodification that can enhance the selected sequence for its intendedtarget.

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to an O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as but not limitedto antisense and RNA interference as these mechanisms require thebinding of a synthetic oligonucleotide strand to an RNA target strand.In the case of antisense, effective inhibition of the mRNA requires thatthe antisense DNA have a very high binding affinity with the mRNA.Otherwise the desired interaction between the synthetic oligonucleotidestrand and target mRNA strand will occur infrequently, resulting indecreased efficacy.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituentalso have been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotideshaving the 2′-MOE modification displayed improved RNA affinity andhigher nuclease resistance. Chimeric oligonucleotides having 2′-MOEsubstituents in the wing nucleosides and an internal region ofdeoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotideor gapmer) have shown effective reduction in the growth of tumors inanimal models at low doses. 2′-MOE substituted oligonucleotides havealso shown outstanding promise as antisense agents in several diseasestates. One such MOE substituted oligonucleotide is presently beinginvestigated in clinical trials for the treatment of CMV retinitis.

In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate. Phosphate protecting groupsinclude those described in U.S. Pat. No. U.S. Pat. No. 5,760,209, U.S.Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475,U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No.6,121,437, U.S. Pat. No. 6,465,628 each of which is expresslyincorporated herein by reference in its entirety.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA:Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesisas appropriate. In addition specific protocols for the synthesis ofoligomeric compounds of the invention are illustrated in the examplesbelow.

Oligonucleotides are generally prepared either in solution or on asupport medium, e.g. a solid support medium. In general a first synthon(e.g. a monomer, such as a nucleoside) is first attached to a supportmedium, and the oligonucleotide is then synthesized by sequentiallycoupling monomers to the support-bound synthon. This iterativeelongation eventually results in a final oligomeric compound or otherpolymer such as a polypeptide. Suitable support medium can be soluble orinsoluble, or may possess variable solubility in different solvents toallow the growing support bound polymer to be either in or out ofsolution as desired. Traditional support medium such as solid supportmedia are for the most part insoluble and are routinely placed inreaction vessels while reagents and solvents react with and/or wash thegrowing chain until the oligomer has reached the target length, afterwhich it is cleaved from the support and, if necessary further worked upto produce the final polymeric compound. More recent approaches haveintroduced soluble supports including soluble polymer supports to allowprecipitating and dissolving the iteratively synthesized product atdesired points in the synthesis (Gravert et al., Chem. Rev., 1997, 97,489-510).

The term support medium is intended to include all forms of supportknown to one of ordinary skill in the art for the synthesis ofoligomeric compounds and related compounds such as peptides. Somerepresentative support medium that are amenable to the methods of thepresent invention include but are not limited to the following:controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g.,Alul, et al., Nucleic Acids Research 1991, 19, 1527); silica-containingparticles, such as porous glass beads and silica gel such as that formedby the reaction of trichloro-[3-(4-chloromethyl)phenyl]propylsilane andporous glass beads (see Parr and Grohmann, Angew. Chem. Internal. Ed.1972, 11, 314, sold under the trademark “PORASIL E” by WatersAssociates, Framingham, Mass., USA); the mono ester of1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, TetrahedronLett., 1970, 4503, sold under the trademark “BIOPAK” by WatersAssociates); TENTAGEL (see, e.g., Wright, et al., Tetrahedron Letters1993, 34, 3373); cross-linked styrene/divinylbenzene copolymer beadedmatrix or POROS, a copolymer of polystyrene/divinylbenzene (availablefrom Perceptive Biosystems); soluble support medium, polyethylene glycolPEG's (see Bonora et al., Organic Process Research & Development, 2000,4, 225-231).

Further support medium amenable to the present invention include withoutlimitation PEPS support a polyethylene (PE) film with pendant long-chainpolystyrene (PS) grafts (molecular weight on the order of 10⁶, (seeBerg, et al., J. Am. Chem. Soc., 1989, 111, 8024 and InternationalPatent Application WO 90/02749),). The loading capacity of the film isas high as that of a beaded matrix with the additional flexibility toaccommodate multiple syntheses simultaneously. The PEPS film may befashioned in the form of discrete, labeled sheets, each serving as anindividual compartment. During all the identical steps of the syntheticcycles, the sheets are kept together in a single reaction vessel topermit concurrent preparation of a multitude of peptides at a rate closeto that of a single peptide by conventional methods. Also, experimentswith other geometries of the PEPS polymer such as, for example,non-woven felt, knitted net, sticks or microwellplates have notindicated any limitations of the synthetic efficacy.

Further support medium amenable to the present invention include withoutlimitation particles based upon copolymers of dimethylacrylamidecross-linked with N,N′-bisacryloylethylenediamine, including a knownamount ofN-tertbutoxycarbonyl-beta-alanyl-N′-acryloylhexamethylenediamine.Several spacer molecules are typically added via the beta alanyl group,followed thereafter by the amino acid residue subunits. Also, the betaalanyl-containing monomer can be replaced with an acryloyl safcosinemonomer during polymerization to form resin beads. The polymerization isfollowed by reaction of the beads with ethylenediamine to form resinparticles that contain primary amines as the covalently linkedfunctionality. The polyacrylamide-based supports are relatively morehydrophilic than are the polystyrene-based supports and are usually usedwith polar aprotic solvents including dimethylformamide,dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, etal., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, andJ. C. S. Perkin I 538 (1981)).

Further support medium amenable to the present invention include withoutlimitation a composite of a resin and another material that is alsosubstantially inert to the organic synthesis reaction conditionsemployed. One exemplary composite (see Scott, et al., J. Chrom. Sci.,1971, 9, 577) utilizes glass particles coated with a hydrophobic,cross-linked styrene polymer containing reactive chloromethyl groups,and is supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA.Another exemplary composite contains a core of fluorinated ethylenepolymer onto which has been grafted polystyrene (see Kent andMerrifield, Israel J. Chem. 1978, 17, 243 and van Rietschoten inPeptides 1974, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp.113-116). Contiguous solid support media other than PEPS, such as cottonsheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) andhydroxypropylacrylate-coated polypropylene membranes (Daniels, et al.,Tetrahedron Lett. 1989, 4345). Acrylic acid-grafted polyethylene-rodsand 96-microtiter wells to immobilize the growing peptide chains and toperform the compartmentalized synthesis. (Geysen, et al., Proc. Natl.Acad. Sci. USA, 1984, 81, 3998). A “tea bag” containingtraditionally-used polymer beads. (Houghten, Proc. Natl. Acad. Sci. USA,1985, 82, 5131). Simultaneous use of two different supports withdifferent densities (Tregear, Chemistry and Biology of Peptides, J.Meienhofer, ed., Ann Arbor Sci. Publ., Ann Arbor, 1972 pp. 175-178).Combining of reaction vessels via a manifold (Gorman, Anal. Biochem.,1984, 136, 397). Multicolumn solid-phase synthesis (e.g., Krchnak, etal., Int. J. Peptide Protein Res., 1989, 33, 209), and Holm and Meldal,in “Proceedings of the 20th European Peptide Symposium”, G. Jung and E.Bayer, eds., Walter de Gruyter & Co., Berlin, 1989 pp. 208-210).Cellulose paper (Eichler, et al., Collect. Czech. Chem. Commun., 1989,54, 1746). Support mediumted synthesis of peptides have also beenreported (see, Synthetic Peptides. A User's Guide, Gregory A. Grant, Ed.Oxford University Press 1992; U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re-34,069.)

Support bound oligonucleotide synthesis relies on sequential addition ofnucleotides to one end of a growing chain. Typically, a first nucleoside(having protecting groups on any exocyclic amine functionalitiespresent) is attached to an appropriate glass bead support andnucleotides bearing the appropriate activated phosphite moiety, i.e. an“activated phosphorous group” (typically nucleotide phosphoramidites,also bearing appropriate protecting groups) are added stepwise toelongate the growing oligonucleotide. Additional methods for solid-phasesynthesis may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat.Nos. 4,725,677 and Re. 34,069.

Commercially available equipment routinely used for the support mediumbased synthesis of oligomeric compounds and related compounds is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. Suitable solid phasetechniques, including automated synthesis techniques, are described inF. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach,Oxford University Press, New York (1991).

The term “solid support tether,” as used herein is generally adi-functional group, covalently binds the ultimate 3′-nucleoside (andthus the nascent oligonucleotide) to the solid support medium duringsynthesis, but which is cleaved under conditions orthogonal to theconditions under which the 5′-protecting group, and if applicable any2′-protecting group, are removed. Suitable solid support tethersinclude, but are not limited to, a divalent group such as alkylene,cycloalkylene, arylene, heterocyclyl, heteroarylene, and the othervariables are as described above. Exemplary alkylene tethers include,but are not limited to, C₁-C₁₂ alkylene (e.g. preferably methylene,ethylene (e.g. ethyl-1,2-ene), propylene (e.g. propyl-1,2-ene,propyl-1,3-ene), butylene, (e.g. butyl-1,4-ene, 2-methylpropyl-1,3-ene),pentylene, hexylene, heptylene, octylene, decylene, dodecylene), etc.Exemplary cycloalkylene groups include C₃-C₁₂ cycloalkylene groups, suchas cyclopropylene, cyclobutylene, cyclopentanyl-1,3-ene,cyclohexyl-1,4-ene, etc. Exemplary arylene tethers include, but are notlimited to, mono- or bicyclic arylene groups having from 6 to about 14carbon atoms, e.g. phenyl-1,2-ene, naphthyl-1,6-ene, napthyl-2,7-ene,anthracenyl, etc. Exemplary heterocyclyl groups within the scope of theinvention include mono- or bicyclic aryl groups having from about 4 toabout 12 carbon atoms and about 1 to about 4 hetero atoms, such as N, Oand S, where the cyclic moieties may be partially dehydrogenated.Certain heteroaryl groups that may be mentioned as being within thescope of the invention include: pyrrolidinyl, piperidinyl (e.g.2,5-piperidinyl, 3,5-piperidinyl), piperazinyl, tetrahydrothiophenyl,tetrahydrofuranyl, tetrahydro quinolinyl, tetrahydro isoquinolinyl,tetrahydroquinazolinyl, tetrahydroquinoxalinyl, etc. Exemplaryheteroarylene groups include mono- or bicyclic aryl groups having fromabout 4 to about 12 carbon atoms and about 1 to about 4 hetero atoms,such as N, O and S. Certain heteroaryl groups that may be mentioned asbeing within the scope of the invention include: pyridylene (e.g.pyridyl-2,5-ene, pyridyl-3,5-ene), pyrimidinyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, etc.

Suitable reagents for introducing the group HOCO-Q-CO include diacids(HO₂C-Q-CO₂H). Particularly suitable diacids include malonic acid (Q ismethylene), succinic acid (Q is 1,2-ethylene), glutaric acid, adipicacid, pimelic acid, and phthalic acid. Other suitable reagents forintroducing HOCO-Q-CO above include diacid anhydrides. Particularlysuitable diacid anhydrides include malonic anhydride, succinicanhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, andphthalic anhydride. Other suitable reagents for introducing HOCO-Q-COinclude diacid esters, diacid halides, etc. One especially preferredreagent for introducing HOCO-Q-CO is succinic anhydride.

The compound of formula may be linked to a support via terminalcarboxylic acid of the HOCO-Q-CO group, via a reactive group on thesupport medium. In some embodiments, the terminal carboxylic acid formsan amide linkage with an amine reagent on the support surface. In otherembodiments, the terminal carboxylic acid forms an ester with an OHgroup on the support medium. In some embodiments, the terminalcarboxylic acid may be replaced with a terminal acid halide, acid ester,acid anhydride, etc. Specific acid halides include carboxylic chlorides,bromides and iodides. Specific esters include methyl, ethyl, and otherC₁-C₁₀ alkyl esters. Specific anhydrides include formyl, acetyl,propanoyl, and other C₁-C₁₀ alkanoyl esters.

The present invention also encompasses the preparation of oligomericcompounds incorporating at least one 2′-O-protected nucleoside into theoligomeric compounds delineated herein. After incorporation andappropriate deprotection the 2′-O-protected nucleoside will be convertedto a ribonucleoside at the position of incorporation. The number andposition of the 2-ribonucleoside units in the final oligomeric compoundcan vary from one at any site or the strategy can be used to prepare upto a full 2′-OH modified oligomeric compound. All 2′-O— protectinggroups amenable to the synthesis of oligomeric compounds are included inthe present invention. In general, a protected nucleoside is attached toa solid support by for example a succinate linker. Then theoligonucleotide is elongated by repeated cycles of deprotecting the5′-terminal hydroxyl group, coupling of a further nucleoside unit,capping and oxidation (alternatively sulfurization). In a morefrequently used method of synthesis the completed oligonucleotide iscleaved from the solid support with the removal of phosphate protectinggroups and exocyclic amino protecting groups by treatment with anammonia solution. Then a further deprotection step is normally requiredfor the more specialized protecting groups used for the protection of2′-hydroxyl groups which will give the fully deprotectedoligonucleotide.

A large number of 2′-O-protecting groups have been used for thesynthesis of oligoribonucleotides but over the years more effectivegroups have been discovered. The key to an effective 2′-O-protectinggroup is that it is capable of selectively being introduced at the2′-O-position and that it can be removed easily after synthesis withoutthe formation of unwanted side products. The protecting group also needsto be inert to the normal deprotecting, coupling, and capping stepsrequired for oligoribonucleotide synthesis. Some of the protectinggroups used initially for oligoribonucleotide synthesis includedtetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groupsare not compatible with all 5′-O-protecting groups so modified versionswere used with 5′-DMT groups such as1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identifieda number of piperidine derivatives (like Fpmp) that are useful in thesynthesis of oligoribonucleotides including1-[(chloro-4-methyl)phenyl]4′-methoxypiperidin-4-yl (Reese et al.,Tetrahedron Lett., 1986, (27), 2291). Another approach was to replacethe standard 5′-DMT (dimethoxytrityl) group with protecting groups thatwere removed under non-acidic conditions such as levulinyl and9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile2′-protecting groups for oligoribonucleotide synthesis. Another morewidely used protecting group initially used for the synthesis ofoligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al.,Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett.,1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The2′-O-protecting groups can require special reagents for their removalsuch as for example the t-butyldimethylsilyl group is normally removedafter all other cleaving/deprotecting steps by treatment of theoligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups(Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoridelabile and photolabile protecting groups that are removed using moderateconditions. One photolabile group that was examined was the[2-(nitrobenzyl)oxy]methyl (nbm) protecting group (Schwartz et al.,Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examinedincluded a number structurally related formaldehyde acetal-derived,2′-O-protecting groups. Also prepared were a number of relatedprotecting groups for preparing 2′-O-alkylated nucleosidephosphoramidites including 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was preparedto be used orthogonally to the TOM group was2′-O—[(R)-1-(2-nitrophenyl)ethyloxy)methyl] ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acidlabile) and an acid labile 2′-O-protecting group has been reported(Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number ofpossible silyl ethers were examined for 5′-O-protection and a number ofacetals and orthoesters were examined for 2′-O-protection. Theprotection scheme that gave the best results was 5′-O-silyl ether-2′-ACE(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether(DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses amodified phosphoramidite synthesis approach in that some differentreagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis ofoligoribonucleotides the main RNA synthesis strategies that arepresently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS), 5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O-[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. Such an activator would also be amenable to the presentinvention.

The primary groups being used for commercial RNA synthesis are:

-   -   TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;    -   TOM=2′-O-[(triisopropylsilyl)oxy]methyl;    -   DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl        ether-2′-O-bis(2-acetoxyethoxy)methyl    -   FPMP=5′-O-DMT-2′-O-[1 (2-fluorophenyl)₄-methoxypiperidin-4-yl].

All of the aforementioned RNA synthesis strategies are amenable to thepresent invention. Strategies that would be a hybrid of the above e.g.using a 5′-protecting group from one strategy with a 2′-O-protectingfrom another strategy is also amenable to the present invention.

Targets of the Invention

“Targeting” an antisense oligomeric compound to a particular nucleicacid molecule, in the context of this invention, can be a multistepprocess. The process usually begins with the identification of a targetnucleic acid whose function is to be modulated. This target nucleic acidmay be, for example, a cellular gene (or mRNA transcribed from the gene)whose expression is associated with a particular disorder or diseasestate, or a nucleic acid molecule from an infectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid. The terms region,segment, and site can also be used to describe an oligomeric compound ofthe invention such as for example a gapped oligomeric compound having 3separate segments.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding a nucleic acid target, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense oligomeric compounds of thepresent invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, oneregion is the intragenic region encompassing the translation initiationor termination codon of the open reading frame (ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsosuitable to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also suitable target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense oligomeric compounds targeted to,for example, DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequences.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso suitable target nucleic acids.

The locations on the target nucleic acid to which the antisenseoligomeric compounds hybridize are hereinbelow referred to as “suitabletarget segments.” As used herein the term “suitable target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense oligomeric compound is targeted. While not wishingto be bound by theory, it is presently believed that these targetsegments represent portions of the target nucleic acid which areaccessible for hybridization.

Exemplary antisense oligomeric compounds include oligomeric compoundsthat comprise at least the 8 consecutive nucleobases from the5′-terminus of a targeted nucleic acid e.g. a cellular gene or mRNAtranscribed from the gene (the remaining nucleobases being a consecutivestretch of the same oligonucleotide beginning immediately upstream ofthe 5′-terminus of the antisense compound which is specificallyhybridizable to the target nucleic acid and continuing until theoligonucleotide contains from about 8 to about 80 nucleobases).Similarly, antisense oligomeric compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds (the remaining nucleobases being a consecutive stretch of thesame oligonucleotide beginning immediately downstream of the 3′-terminusof the antisense compound which is specifically hybridizable to thetarget nucleic acid and continuing until the oligonucleotide containsfrom about 8 to about 80 nucleobases). One having skill in the art armedwith the antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further antisense compounds.

Once one or more target regions, segments or sites have been identified,antisense oligomeric compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

In accordance with one embodiment of the present invention, a series ofnucleic acid duplexes comprising the antisense oligomeric compounds ofthe present invention and their complements can be designed for aspecific target or targets. The ends of the strands may be modified bythe addition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the duplex is then designed andsynthesized as the complement of the antisense strand and may alsocontain modifications or additions to either terminus. For example, inone embodiment, both strands of the duplex would be complementary overthe central nucleobases, each having overhangs at one or both termini.

For example, a duplex comprising an antisense oligomeric compound havingthe sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:1) and having atwo-nucleobase overhang of deoxythymidine(dT) would have the followingstructure:    cgagaggcggacgggaccgdTdT Antisense Strand (SEQ ID NO:2)   ||||||||||||||||||| dTdTgctctccgcctgccctggc Complement Strand (SEQ IDNO:3)

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from various RNA synthesis companies such as for exampleDharmacon Research Inc., (Lafayette, Colo.). Once synthesized, thecomplementary strands are annealed. The single strands are aliquoted anddiluted to a concentration of 50 uM. Once diluted, 30 uL of each strandis combined with 15 uL of a 5× solution of annealing buffer. The finalconcentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. Thissolution is incubated for 1 minute at 90° C. and then centrifuged for 15seconds. The tube is allowed to sit for 1 hour at 37° C. at which timethe dsRNA duplexes are used in experimentation. The final concentrationof the dsRNA compound is 20 uM. This solution can be stored frozen (−20°C.) and freeze-thawed up to 5 times.

Once prepared, the desired synthetic duplexs are evaluated for theirability to modulate target expression. When cells reach 80% confluency,they are treated with synthetic duplexs comprising at least oneoligomeric compound of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired dsRNA compound at a finalconcentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

In a further embodiment, the “suitable target segments” identifiedherein may be employed in a screen for additional oligomeric compoundsthat modulate the expression of a target. “Modulators” are thoseoligomeric compounds that decrease or increase the expression of anucleic acid molecule encoding a target and which comprise at least an8-nucleobase portion which is complementary to a suitable targetsegment. The screening method comprises the steps of contacting asuitable target segment of a nucleic acid molecule encoding a targetwith one or more candidate modulators, and selecting for one or morecandidate modulators which decrease or increase the expression of anucleic acid molecule encoding a target. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding a target, the modulator may then be employed in furtherinvestigative studies of the function of a target, or for use as aresearch, diagnostic, or therapeutic agent in accordance with thepresent invention.

The suitable target segments of the present invention may also becombined with their respective complementary antisense oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides.

Hybridization

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,one mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An antisense oligomeric compound is specifically hybridizable whenbinding of the compound to the target nucleic acid interferes with thenormal function of the target nucleic acid to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense oligomeric compound to non-targetnucleic acid sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and under conditions in which assaysare performed in the case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention, “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases regardless of where the two are located. Forexample, if a nucleobase at a certain position of an oligomeric compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, the target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleobases which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleobases such that stable and specificbinding occurs between the oligonucleotide and a target nucleic acid.

It is understood in the art that the sequence of an antisense oligomericcompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). The antisense oligomeric compounds ofthe present invention can comprise at least about 70%, at least about80%, at least about 90%, at least about 95%, or at least about 99%sequence complementarity to a target region within the target nucleicacid sequence to which they are targeted. For example, an antisenseoligomeric compound in which 18 of 20 nucleobases of the antisenseoligomeric compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense oligomeric compound which is 18nucleobases in length having 4 (four) noncomplementary nucleobases whichare flanked by two regions of complete complementarity with the targetnucleic acid would have 77.8% overall complementarity with the targetnucleic acid and would thus fall within the scope of the presentinvention. Percent complementarity of an antisense oligomeric compoundwith a region of a target nucleic acid can be determined routinely usingBLAST programs (basic local alignment search tools) and PowerBLASTprograms known in the art (Altschul et al., J. Mol. Biol., 1990, 215,403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

Screening and Target Validation

In some embodiments, “suitable target segments” may be employed in ascreen for additional oligomeric compounds that modulate the expressionof a selected protein. “Modulators” are those oligomeric compounds thatdecrease or increase the expression of a nucleic acid molecule encodinga protein and which comprise at least an 8-nucleobase portion which iscomplementary to a suitable target segment. The screening methodcomprises the steps of contacting a suitable target segment of a nucleicacid molecule encoding a protein with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding a protein.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a peptide, the modulator may then beemployed in further investigative studies of the function of thepeptide, or for use as a research, diagnostic, or therapeutic agent inaccordance with the present invention.

The suitable target segments of the present invention may also becombined with their respective complementary antisense oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides. Such double stranded oligonucleotidemoieties have been shown in the art to modulate target expression andregulate translation as well as RNA processsing via an antisensemechanism. Moreover, the double-stranded moieties may be subject tochemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmonsand Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al.,Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., GenesDev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498;Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target (Tijsterman etal., Science, 2002, 295, 694-697).

The oligomeric compounds of the present invention can also be applied inthe areas of drug discovery and target validation. The present inventioncomprehends the use of the oligomeric compounds and targets identifiedherein in drug discovery efforts to elucidate relationships that existbetween proteins and a disease state, phenotype, or condition. Thesemethods include detecting or modulating a target peptide comprisingcontacting a sample, tissue, cell, or organism with the oligomericcompounds of the present invention, measuring the nucleic acid orprotein level of the target and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furtheroligomeric compound of the invention. These methods can also beperformed in parallel or in combination with other experiments todetermine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature (2001), 411,494-498; Nishikura et al., Cell (2001), 107, 415-416; and Bass et al.,Cell (2000), 101, 235-238.)

Kits, Research Reagents, Diagnostics, and Therapeutics

The oligomeric compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the oligomeric compounds of the presentinvention, either alone or in combination with other oligomericcompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense oligomeric compounds are compared tocontrol cells or tissues not treated with antisense oligomeric compoundsand the patterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds and or oligomeric compounds which affectexpression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The oligomeric compounds of the invention are useful for research anddiagnostics, because these oligomeric compounds hybridize to nucleicacids encoding proteins. For example, oligonucleotides that are shown tohybridize with such efficiency and under such conditions as disclosedherein as to be effective protein inhibitors will also be effectiveprimers or probes under conditions favoring gene amplification ordetection, respectively. These primers and probes are useful in methodsrequiring the specific detection of nucleic acid molecules encodingproteins and in the amplification of the nucleic acid molecules fordetection or for use in further studies. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid can be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofselected proteins in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligomeric compoundshave been employed as therapeutic moieties in the treatment of diseasestates in animals, including humans. Antisense oligonucleotide drugs,including ribozymes, have been safely and effectively administered tohumans and numerous clinical trials are presently underway. It is thusestablished that antisense oligomeric compounds can be usefultherapeutic modalities that can be configured to be useful in treatmentregimes for the treatment of cells, tissues and animals, especiallyhumans.

For therapeutics, an animal, such as a human, suspected of having adisease or disorder which can be treated by modulating the expression ofa selected protein is treated by administering antisense oligomericcompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a protein inhibitor. The protein inhibitors of the present inventioneffectively inhibit the activity of the protein or inhibit theexpression of the protein. In some embodiments, the activity orexpression of a protein in an animal or cell is inhibited by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 99%,or by 100%.

For example, the reduction of the expression of a protein may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. The cells contained within the fluids, tissuesor organs being analyzed can contain a nucleic acid molecule encoding aprotein and/or the protein itself.

The oligomeric compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of anoligomeric compound to a suitable pharmaceutically acceptable diluent orcarrier. Use of the oligomeric compounds and methods of the inventionmay also be useful prophylactically.

In another embodiment, the present invention provides for the use of acompound(s) of the invention in the manufacture of a medicament for thetreatment of any and all diseases and conditions disclosed herein.

Formulations

The antisense oligomeric compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the oligomeric compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligomeric compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

EXAMPLES

The compounds and processes of the present invention will be describedfurther in detail with respect to specific preferred embodiments by wayof examples, it being understood that these are intended to beillustrative only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1

Synthesis of Peptide Nucleic Acid (PNA)-Peptide Conjugates Containing anActivated Disulfide.

The PNA part of the conjugates was assembled using an automated 433 Apeptide synthesizer (Applied Biosystems) and commercially availabletert-butyloxycarbonyl/benzyloxycarbonyl (Boc/Cbz) protected PNA monomers(Applied Biosystems) according to previously published procedures forPNA synthesis (Boc chemistry) (See L. Christensen, et al. (1995), J.Pept. Sci. 1, 175-183; T. Koch, et al. (1997), J. Pept. Res. 49,80-88.). The synthesis was performed on MBHA LL polystyrene resin(NovaBiochem), pre-loaded to about 0.1-0.2 mmol/g with eitherBoc-Lys(2-Cl-Z)-OH (NovaBiochem) for N-terminal modification orBoc-Lys(Fmoc)-OH (NovaBiochem) for modification of the C-terminal Lys.

The synthesis of the peptide part of the conjugate was carried out bycontinuing the synthesis on the N-terminus of the PNA according tostandard procedures for solid phase peptide synthesis. The thiolfunctionality was introduced according to standard procedures for solidphase peptide synthesis using N-acetyl-Cys(Trt)-OH or any othercarboxylic acid containing a suitably protected thiol after removingeither the Fmoc group at the C-terminal Lys or the Boc group of theN-terminus of the PNA-peptide conjugate. For final deprotection andcleavage one vol. of TFA/TFMSA/thioanisol/m-cresol (8:1:1:1) was addedand the suspension was shaken for 1.5-6 h. The filtrate was then addedto a 10-fold volume of cold diethylether, mixed and centrifuged. Thesupernatant was removed and the pellet was re-suspended in ether. Thiswas repeated three times. The pellet was dried and re-dissolved indegassed water/pyridine (8:2) before 10 equiv. of2,2-dipyridyl-disulfide in acetonitrile (ca. 0.02-0.1 M) was added toform the activated disulfide (Scheme 1). The pH of the mixture wasadjusted to pH 8.5-9.0 with aqueous TEA and the mixture was shaken forca. 3-6 h. After evaporation of the solvents in vacuo, the residue wasdissolved in water and washed with ether to remove the excessdithiodipyridine. After ether removal, purification was performed on aGilson HPLC system (215 liquid handler, 155 UV/VIS and 321 pump), byreverse phase high performance liquid chromatography (RP-HPLC), using aZorbax (C-3, 5 μm, 300 Å, 250×7.8 mm, 4 mL/min). A linear gradient fromsolvent A: 0.1% heptafluorobutyric acid in water to B: acetonitrile wasused as the liquid phase. Product-containing fractions were combined andpurity was determined by analytical HPLC and composition confirmed byelectrospray mass spectrometry. Samples were lyophilized and stored at−20° C. until before use. Representative examples of PNAs, which carrythe dithiopyridine functionality at their C- or their N-terminus, arelisted in Table 1. TABLE 1 Examples of PNAs containing the activateddisulfide functionality. ISIS # Sequence MW_(calc) MW_(ob) 347349KKOrnKKOrnKK-CACAGATGACATTAG- 5620 5617 K(R¹) 350126(dK)₈-CACAGATGACATTAG-K(R²) 5675 5673 350226 (dK)₈-CACAGATGACATTAG-K(R¹)5647 5645 358346 R¹-(dK)₈-CACAGATGACATTAG-K 5620 5617 368640R¹-(dK)₈-CACAGATGACATTAG-K(Bio) 6019 6017Where: Orn = L-ornithine and dK = D-lysine

Example 2

Synthesis of ISIS367745

The oligonucleotide was assembled using phosphoramidite chemistryaccording to standard procedures known to one of ordinary skill in theart for solid phase synthesis of oligonucleotides. A suitably protecteddisulfide containing phosphoramidite was coupled to the 5′-end of theoligonucleotide on resin, using standard phosphoramidite chemistry (seeabove scheme).

After final deprotection and cleavage from the resin, thedisulfide-modified oligonucleotide was purified by reversed phase HPLCand the thiol was generated by adding a 0.1 M aqueous solution of DTTaccording to manufacturer's protocol. The oligonucleotide was passedthrough a size exclusion column (Sephadex G25) to separate from excessDTT and the product-containing fractions were combined and evaporated.The oligonucleotide was then re-dissolved in degassed water/pyridine(80:20) and a solution of 10 equiv. 2,2-dipyridyl-disulfide inacetonitrile (ca. 0.02-0.1 M) was added. The pH of the mixture wasadjusted to pH 8.5-9.0 with aqueous TEA and the mixture was shaken forca. 3-6 h. After evaporation of the solvents in vacuo, the residue wasdissolved in water and purified by size exclusion chromatography(Sephadex G25) followed by reversed phase HPLC. The product-containingfractions were combined and evaporated in vacuo and the compound wasstored at −20° C. until use.

ISIS367745—an Oligonucleotide Containing an Activated Disulfide at the5′-End Isis # Sequence/Modification MW_(calc) MW_(ob) 367745X-CTGCTAGCCTCTGGATTTGAall PS; _ = 2′-MOE 7521

Example 3

Synthesis of Oligonucleotides Containing an Activated Disulfide at the5′-End.

The oligonucleotide is assembled using phosphoramidite chemistryaccording to standard procedures known to one of skill in the art forsolid phase synthesis of oligonucleotides. At the 5′-end, a suitablyprotected monomer (see below for non-limiting examples) is coupled tothe oligonucleotide on the resin using phosphoramidite chemistry.

Exemplified Phosphoramidite Building Blocks for Introducing an ActivatedDisulfide at the 5′-Terminus of an Oligonucleotide.

where examples of X include, but are not limited to

R=thiol protecting group, or S-disulfide protecting groupAfter final deprotection and cleavage from the resin, the modifiedoligonucleotide is purified by reversed phase HPLC and the thiolgenerated using relevant, standard deprotection conditions. Thethiol-modified oligonucleotide is purified and dissolved in degassedwater/pyridine (80:20) to which a solution of 10 equiv.2,2-dipyridyl-disulfide in acetonitrile (ca. 0.02-0.1 M) is added. ThepH of the mixture is adjusted to pH 8.5-9.0 with aqueous TEA and themixture is shaken for ca. 3-6 h. After evaporation of the solvents invacuo, the residue is dissolved in water and purified by size exclusionchromatography (Sephadex G25) followed by reversed phase HPLC. Theproduct-containing fractions are combined and evaporated in vacuo andthe compound is stored at −20° C. until use.

Example 4

Synthesis of Oligonucleotides Containing an Activated Disulfide at the3′-End.

A solid support is bound to a spacer group via its free hydroxyl usingstandard ester formation chemistry, known to one of ordinary skill inthe art. The spacer group is comprised of a free hydroxyl, a protectedhydroxyl, and a protected sulfide or a protected disulfide. Theprotected hydroxyl is deprotected and the oligonucleotide is assembledthereon using phosphoramidite chemistry according to standard proceduresknown to one of ordinary skill in the art in solid phase synthesis ofoligonucleotides. After the oligonucleotide has been fully synthesized,spacer-bound or thiol-modified oligonucleotide, is deprotected, cleavedand finally purified by reversed phase HPLC. The thiol is generatedusing relevant, standard thiol deprotection conditions. Thethiol-modified oligonucleotide is purified and dissolved in degassedwater/pyridine (80:20) to which a solution of 10 equiv.2,2-dipyridyl-disulfide in acetonitrile (ca. 0.02-0.1 M) is added. ThepH of the mixture is adjusted to pH 8.5-9.0 with aqueous TEA and themixture is shaken for ca. 3-6 h. After evaporation of the solvents invacuo, the residue is dissolved in water and purified by size exclusionchromatography (Sephadex G25) followed by reversed phase HPLC. Theproduct-containing fractions are combined and evaporated in vacuo andthe compound is stored at −20° C. until use. Examples of spacers whichare useful in synthesizing the thiol-modfied oligonucleotides of thepresent invention:

Example 5

Synthesis of Oligonucleotides Containing an Activated Disulfide at the2′ Position.

The oligonucleotide is assembled using phosphoramidite chemistryaccording to standard procedures known to one of skill in the art forsolid phase synthesis of oligonucleotides. A protected thiol ordisulfide is introduced within the sequence using a nucleosidephosphoramidite modified at the 2′-position with a suitably protectedthiol or disulfide functionality, as indicated below.

where examples of X include, but are not limited to

R=thiol protecting group, or S-disulfide protecting groupAfter final deprotection and cleavage from the resin, the-modifiedoligonucleotide is purified by reversed phase HPLC and the thiolgenerated using relevant, standard deprotection conditions. Thethiol-modified oligonucleotide is purified and dissolved in degassedwater/pyridine (80:20) to which a solution of 10 equiv.2,2-dipyridyl-disulfide in acetonitrile (ca. 0.02-0.1 M) is added. ThepH of the mixture is adjusted to pH 8.5-9.0 with aqueous TEA and themixture is shaken for ca. 3-6 h. After evaporation of the solvents invacuo, the residue is dissolved in water and purified by size exclusionchromatography (Sephadex G25) followed by reversed phase HPLC. Theproduct-containing fractions are combined and evaporated in vacuo andthe compound is stored at −20° C. until use.

Example 6

Synthesis of siRNA Duplexes Containing Activated Disulfides.

The two complementary sense and antisense strands composed of natural ormodified ribonucleotide building blocks are assembled usingphosphoramidite chemistry according to standard procedures known to oneof skill in the art for solid phase synthesis of oligonucleotides.Activated disulfide groups are incorporated into the oligonucleotidestrands, as described herein in examples 3, 4 or 5. Prior to use, theactivated disulfide modified oligonucleotide is annealed to itscomplementary unmodified strand to form the siRNA duplex by mixing equalamounts of both compounds in water or physiological buffer, heating themixture for ca. 10 min to ca. 90° C. and slowly cooling the mixture tor.t.

Example 7

Protein Binding of Oligomers in Mouse Plasma.

Mouse plasma solutions were prepared by adding 12 μL of a 1 mM aqueoussolution of oligomer containing activated disulfides to 1.2 mL of 100%mouse plasma stabilized with 10 mM Hepes. Prior to addition of oligomer,glutathione (GSH:GSSG, 50:1) was added to one of the samples to a finalconcentration of 10 μM. The samples were incubated at 37° C. for 24.6 hand aliquots (100 μL) were removed for analysis at 0.1, 0.25, 0.5, 1.0,2.25, 4.0, 7.5, 24.0 and 24.6 h. At 24.1 h GSH was added to thesolutions to a final conc. of 5 mM and incubated for an additional 0.5h. The last aliquot used for protein binding analysis was removed at24.6 h. For HPLC analysis, 200 μL of 10% dichloroacetic acid in H₂O:AcCN(5:1) was added to each aliquot and the precipitated plasma proteinswere removed by centrifugation. 200 μL of supernatant was carefullyremoved, 12 μL of the standard solution was added (PNA 6-mer, 0.1 mMaqueous solution) and the samples were subjected to HPLC analysis.(Zorbax 300 SB C₃ column at 60° C.; eluent A: 0.1% aq. HFBA, B: CH₃CN;gradient: 0-60 in 30 min.). For further details please see 1) Bellomo,G.; Vairetti, M.; Stivala, L.; Mirabelli, F.; Richelmi, P. et al.Demonstration of nuclear compartmentalization of glutathione inhepatocytes. Proc Natl Acad Sci USA 1992, 89, 4412-4416; or 2)Martinovich, G. G.; Cherenkevich, S. N.; Sauer, H. Intracellular redoxstate: towards quantitative description. Eur Biophys J 2005.

Example 8

Bio-Reversible Protein Binding of Oligomer Containing ActivatedDisulfides in Mouse Plasma.

In one embodiment of the invention ISIS350226, a PNA-peptide conjugate,containing an activated disulfide, was examined for protein binding, asdescribed herein in Example 7. A graphical analysis of the results ofthis experiment is shown in FIG. 2 a. As shown in FIG. 2 a the PNAconjugate remains attached to the plasma proteins for over 24 h. Releaseof the PNA-peptide conjugate from the plasma proteins was facilitated bythe addition of 5 mM glutathione (a concentration similar to that insidea cell) as described herein in Example 7. A graphical analysis of theresult of this experiment is shown in FIG. 2 b. A similar experiment wascarried out where the release of the PNA-peptide conjugate using 5 mMGSH was monitored over time as shown in FIG. 2 c. These data indicatethat the reductive environment inside the cell will release the PNA fromthe plasma proteins. Therefore, an embodiment of the invention is anactivated oligomer which is capable of forming a bio-reversible bondwith plasma proteins, i.e. the activated oligomer is bound to plasmaproteins under plasma conditions, however become released under cellularconditions.

Example 9

Effect of Mouse Plasma Protein Binding on Enzymatic Stability ofOligomeric Compounds.

Oligomeric compounds were incubated with mouse plasma (100%) at 37° C.,at a concentration of 10 μM. At certain time points an aliquot wasremoved from the incubating stock solution. Reduced glutathione (GSH)was immediately added to the aliquot to yield a GSH concentration of 10mM. The aliquot was incubated at 37° C. for 15 min and then added todouble the volume of a 10% dichloroacetic acid solution inwater:acetonitrile (5:1). The precipitate was spun down bycentrifugation and 2/3 of the supernatant was transfered to a test tubefor HPLC analysis. A standard was added to the test tube forquantification purposes. The degradation of the peptide portion of theconjugate was measured as the percent of the full length parent compoundas a function of time by HPLC.

Example 10

Effect of Mouse Plasma Protein Binding on Enzymatic Stability ofOligomers Containing Activated Disulfides.

In one embodiment of the invention, two oligomers, ISIS347349, apeptide-PNA conjugate containing an activated disulfide and ISIS 309145,a peptide-PNA conjugate control containing no activated disulfide, wereexamined for the effect of mouse plasma protein binding on enzymaticstability, as described herein in example 9. Graphical analyses of theresults of these experiments are shown in FIG. 3.

Example 11

Radiolabeling of PNA-peptide conjugate Isis 358346 with³H-acetylchloride.

The PNA-peptide conjugate Isis 358346 was synthesized by Boc chemistryon MBHA resin pre-loaded with Boc-Lys(Fmoc)-OH as described herein inexample 1. Subsequently, the Fmoc-protection was removed from theC-terminal Lys side chain with piperidine in DMF according to standardprocedures. The resin was then washed with DMF and DCM and dried invacuo. Custom radiolabeling of the compounds was carried out at MoravekBiochemicals as follows.The resin was placed into a small custom made synthesis column equippedwith a glass frit, a stop cock and an argon inlet and allowed to swellin DCM for 2 h. After removal of the solvent, a mixture of 5.0 equiv.acetylchloride (methyl, ³H) and 15 equiv. N,N-diisopropylethylamine(DIEA) in 300 μL dry DCM was added and allowed to react for 1 h underfrequent mixing. The resin was washed with DCM, pyridine and again withDCM and a mixture of 2% acetic anhydride in anhydrous pyridine/DMF wasadded to the resin and allowed to react for 10 min under frequentmixing. This step was repeated twice and the resin was washed thoroughlywith pyridine, DMF and DCM. Finally, the compound was deprotected,cleaved from resin, converted to the dithiopyridine derivative andpurified by reversed phase HPLC purification according to the conditionsas described herein in example 1. The purified compound was converted tothe chloride salt using a Dowex anion exchange resin (Dowex 2×8-200,Aldrich 428639 converted to Cl-form). All sample-containing fractionswere combined and evaporated. The sample was finally redissolved in 25%aqueous EtOH and stored frozen until use.

Example 12

PK/Tissue Distribution Studies in Balb/c Mice.

Radiolabeled oligomeric compopunds, either free or pre-bound to mouseplasma, were administered by i.v. tail vein injection into Male Balb/cmice (n=3 per time point per compound) at a dose of 3 mg/kg (0.5 mg/mLin PBS with 3% EtOH, pH 7.1). Mice were sacrificed after 4, 24 and 48hours and samples from blood, feces, urine, heart, lung, liver, kidney,spleen, mesentery lymph nodes, pancreas, small/large intestine, colon,testes, skeleton muscle and furless skin were collected and dissolved intissue solubilizer. The samples were then added to liquid scintillationbuffer and measured in a LSC instrument (Beckman, LS6000IC).

Example 13

PK/Tissue Distribution Studies of Isis 358346 in Balb/c Mice

In one embodiment of the invention, radiolabeled (³H) Isis 358346, apeptide-PNA conjugate containing an activated disulfide, prepared asdescribed herein in example 11 was examined for PK/tissue distribution,as described herein in example 12. Graphical analyses of the results ofthese experiments are shown in FIG. 4.

Example 14

Synthesis of uniform 2′-O-MOE oligonucleotide ISIS 386773 bearing apyridyldisulfide functionality at the 5′-terminus.

The oligonucleotide ISIS 386773 and its unmodified control ISIS 364615listed in the following table were synthesized and purified as describedin Examples 2 and 3. ISIS # Sequence 5′ to 3′ MW_(calc) MW_(found)364615                       TGTCTCTGGTCCTTACTT 6826.1 6826.1 386773Py-S—S—(CH₂)₈—O—P(O)₂-TGTCTCTGGTCCTTACTT 7131.3 7131.2

Example 15

Determination of the Plasma Protein Binding Capacity for ISIS 386773.

ISIS 386773 and its unmodified control ISIS 364615 were dissolved in PBSbuffer and added to 100% mouse plasma to a final concentration rangingfrom 5 to 200 μM. The samples were incubated at 37° C. for ca. 30 minbefore the plasma proteins were quantitatively precipitated by theaddition of 2 volumes of acetonitrile/ethanol (2:1). The samples werecentrifuged in a bench top centrifuge at 13,000 rpm and the supernatantwas analyzed by UV spectroscopy. The absornace at 260 nm was recordedand used to calculate the amount of oligonucleotide in the supernatantas well as the amount oligonucleotide bound. FIG. 5 shows the plasmaprotein binding of the two oligonucleotides as a function of theirplasma concentration. While the ISIS 386773 bearing the activateddisulfide is significantly bound in the concentration rangeinvestigated, no binding could be detected for ISIS 364615, itsunmodified control.

Example 16

Release of ISIS 386773 from Plasma Proteins Under Reductive Conditions.

The kinetics for the release of ISIS 386773 was measured after theaddition of GSH in PBS to a final concentration of 5 mM to a sample ofthe oligonucleotide pre-bound to mouse plasma similar to Example 8.Aliquots were taken after 0, 15 30 and 60 min and plasma proteins wereprecipitated by the addition of 2 volumes of acetonitrile/ethanol (2:1).The samples were centrifuged in a bench top centrifuge at 13,000 rpm andthe supernatant was analyzed by HPLC after the addition of a definedamount of a standard (PNA 6mer: H-CTATAG-NH₂). The amount ofoligonucleotide released was determined by the ratio ofanalyte/standard. A non-GSH-treated sample and an aqueousnon-plasma-containing sample were treated and analyzed identically andused to determine the 100%-bound and 100%-unbound reference levels,respectively. FIG. 6 shows the release kinetics of ISIS 386773 fromplasma proteins after the addition of GSH.

Example 17

Kinetics of Plasma Protein Binding of ISIS 386773.

The kinetics for the binding of ISIS 386773 to plasma proteins wasmeasured in 100% mouse plasma. Aliquots were taken after 1, 2, 4, 8, 16and 32 min and plasma proteins were precipitated by the addition of 2volumes of acetonitrile/ethanol (2:1). The samples were centrifuged in abench top centrifuge at 13,000 rpm and the supernatant was analyzed byHPLC after the addition of a defined amount of a standard (PNA 6mer:H-CTATAG-NH₂) (Zorbax 300 SB C₃ column at 60° C.; eluent A: 0.1 M aq.ammonium acetate, B: CH₃CN; gradient: 0-60 in 30 min.). The amount ofoligonucleotide released was determined by the ratio ofanalyte/standard. An aqueous non-plasma-containing sample was treatedand analyzed identically and used to determine the 100%-unboundreference level. FIG. 7 shows the binding kinetics of ISIS 386773 toplasma proteins.

Although the invention has been described in detail with respect tovarious preferred embodiments it is not intended to be limited thereto,but rather those skilled in the art will recognize that variations andmodifications may be made therein which are within the spirit of theinvention and the scope of the appended claims.

All publications, patents, published patent applications, and otherreferences mentioned herein are hereby incorporated by reference intheir entirety.

1. A method of treating a subject with an activated oligomer, the methodcomprising: a. providing an activated oligomer, said activated oligomerconjugated optionally with a bivalent linking group to an activateddisulfide moiety; and b. administering the activated oligomer to saidsubject.
 2. The method of claim 1, wherein the activated oligomer isselected from an oligonucleotide, a peptide nucleic acid or a morpholinonucleic acid.
 3. The method of claim 2, wherein the activated oligomeris an oligonucleotide.
 4. The method of claim 3, wherein theoligonucleotide comprises at least one 2′-modified nucleotide, whereinsaid 2′-modification is selected from halogen, alkoxy, substitutedalkoxy, amino, or substituted amino.
 5. (canceled)
 6. The method ofclaim 3, wherein the oligonucleotide comprises at least onephosphorothioate internucleoside linkage.
 7. The method of claim 3,wherein the oligonucleotide comprises at least one phosphodiesterinternucleoside linkage.
 8. The method of claim 2, wherein the activatedoligomer is a peptide nucleic acid.
 9. (canceled)
 10. The method ofclaim 2, wherein the activated oligomer is a double-strandedoligonucleotide.
 11. The method of claim 10, wherein the double-strandedoligonucleotide comprises a first strand and a second strand.
 12. Themethod of claim 11, wherein at least one nucleotide of the first or thesecond strand of the double-stranded oligonucleotide is 2′-modifiednucleotide, wherein said 2′modification is selected from halogen,alkoxy, substituted alkoxy, amino, or substituted amino.
 13. (canceled)14. The method of claim 11, wherein at least one internucleoside linkageof the first or the second strand of the double-stranded oligonucleotideis a phophorothioate.
 15. The method of claim 11, wherein at least oneinternucleoside linkage of the first or the second strand of thedouble-stranded oligonucleotide is a phosphodiester.
 16. The method ofclaim 1, wherein the activated disulfide moiety is selected frommoieties of formula —S—S(O)_(n)—R₁, wherein n is 0, 1, or 2; and R₁ is asubstituted or unsubstituted heterocyclic. 17.-20. (canceled)
 18. Themethod of claim 16, wherein n is 0 and R₁ is substituted orunsubstituted pyridyl, substituted or unsubstituted benzothiazolyl, orsubstituted or unsubstituted tetrazolyl.
 19. The method of claim 21,wherein R₁ is selected from 2-pyridyl, 3-nitro-2pyridyl,5-nitro-2-pyridyl, 2-benzothiazolyl, N-(C₁-C₁₂ alkyl)-2-pyridyl,N-oxide-2-pyridyl, or 1-methyl-2-1H-tetrazolyl.
 20. The method of claim1, wherein the bivalent linking group is a bivalent substituted orunsubstituted aliphatic group.
 21. The method of claim 1, wherein thebivalent linking group is of the formula: -Q₁-G-Q₂-, wherein Q₁ and Q₂are independently absent or selected from substituted or unsubstitutedC₁-C₁₂ alkylene, substituted or unsubstituted alkarylene or—(CH₂)_(m)—O—(CH₂)_(p)—, wherein each m and p are, independently, aninteger from 1 to about 10; G is —NH—C(O)—, —C(O)—NH—, —NH—C(O)—NH—,—NH—C(S)—NH—, —NH—O—, NH—C(O)—O—, or —O—CH₂—C(O)—NH—.
 22. The method ofclaim 20, wherein the bivalent linking group is selected from:


23. The method of claim 1, wherein the step of administering isintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion. 24.-115. (canceled)